Spiro 2,4 pyrimidinediamine compounds and their uses

ABSTRACT

The present invention provides methods of treating or preventing autoimmune diseases with spiro 2,4-pyrimidinediamine compounds, as well as methods of treating, preventing or ameliorating symptoms associated with such diseases. Specific examples of autoimmune diseases that can be treated or prevented with the compounds include rheumatoid arthritis and/or its associated symptoms, systemic lupus erythematosis and/or its associated symptoms and multiple sclerosis and/or its associated symptoms.

1. CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. § 119(e) to applicationSer. No. 60/630,808, filed Nov. 24, 2004, the content of which isincorporated herein by reference.

2. FIELD

The present invention relates generally to spiro 2,4-pyrimidinediaminecompounds, pharmaceutical compositions comprising the compounds,intermediates and synthetic methods of making the compounds and methodsof using the compounds and compositions in a variety of contexts, suchas in the treatment or prevention of autoimmune diseases and/or thesymptoms associated therewith.

3. BACKGROUND

Crosslinking of Fc receptors, such as the high affinity receptor for IgE(FcεRI) and/or the high affinity receptor for IgG (FcγRI) activates asignaling cascade in mast, basophil and other immune cells that resultsin the release of chemical mediators responsible for numerous adverseevents. For example, such crosslinking leads to the release of preformedmediators of Type I (immediate) anaphylactic hypersensitivity reactions,such as histamine, from storage sites in granules via degranulation. Italso leads to the synthesis and release of other mediators, includingleukotrienes, prostaglandins and platelet-activating factors (PAFs),that play important roles in inflammatory reactions. Additionalmediators that are synthesized and released upon crosslinking Fcreceptors include cytokines and nitric oxide.

The signaling cascade(s) activated by crosslinking Fc receptors such asFcεRI and/or FcγRI comprises an assay of cellular proteins. Among themost important intracellular signal propagators are the tyrosinekinases. And, an important tyrosine kinase involved in the signaltransduction pathways associated with crosslinking the FcεRI and/orFcγRI receptors, as well as other signal transduction cascades, is Sykkinase (see Valent et al., 2002, Intl. J. Hematol. 75(4):257-362 forreview).

As the mediators released as a result of FcεRI and FcγRI receptorcross-linking are responsible for, or play important roles in, themanifestation of numerous adverse events, the availability of compoundscapable of inhibiting the signaling cascade(s) responsible for theirrelease would be highly desirable. Moreover, owing to the critical rolethat Syk kinase plays these and other receptor signaling cascade(s), theavailability of compounds capable of inhibiting Syk kinase would also behighly desirable.

4. SUMMARY

In one aspect, the present invention provides novel spiro2,4-pyrimidinediamine compounds that, as will be discussed in moredetail below, have myriad biological activities. The compounds generallycomprise a 2,4-pyrimidinediamine “core” having the following structureand numbering convention:

The compounds described herein are substituted at the C2 nitrogen (N2)and C4 nitrogen (N4) to form two secondary amines and are optionallyfurther substituted at one or more of the following positions: the C5position and/or the C6 position. The substituent at N2, as well as theoptional substituents at the other positions, other than N4, may rangebroadly in character and physico-chemical properties. For example, thesubstituent(s) may be a branched, straight-chained or cyclic alkyl, abranched, straight-chained or cyclic heteroalkyl, a mono- or polycyclicaryl a mono- or polycyclic heteroaryl or combinations of these groups.These substituent groups may be further substituted, as will bedescribed in more detail below. The N4 substituent will generallycontain a spiro heterocyclic group as described, infra.

The N2 and/or N4 substituents may be attached directly to theirrespective nitrogen atoms, or they may be spaced away from theirrespective nitrogen atoms via linkers, which may be the same ordifferent. The nature of the linkers can vary widely, and can includevirtually any combination of atoms or groups useful for spacing onemolecular moiety from another. For example, the linker may be an acyclichydrocarbon bridge (e.g., a saturated or unsaturated alkyleno such asmethanol ethano, etheno, propano, prop[1]eno, butano, but[1]eno,but[2]eno, buta[1,3]dieno, and the like), a monocyclic or polycyclichydrocarbon bridge (e.g., [1,2]benzeno, [2,3]naphthaleno, and the like),a simple acyclic heteroatomic or heteroalkyldiyl bridge (e.g., —O—, —S—,—S—O—, —NH—, —PH—, —C(O)—, —C(O)NH—, —S(O)—, —S(O)₂—, —S(O)NH—,—S(O)₂NH—, —O—CH₂—, —CH₂—O—CH₂—, —O—CH═CH—CH₂—, and the like), amonocyclic or polycyclic heteroaryl bridge (e.g., [3,4]furano, pyridino,thiopheno, piperidino, piperazino, pyrazidino, pyrrolidino, and thelike) or combinations of such bridges.

The substituents at the N2, N4, C5 and/or C6 positions, as well as theoptional linkers, may be further substituted with one or more of thesame or different substituent groups. The nature of these substituentgroups may vary broadly. Non-limiting examples of suitable substituentgroups include branched, straight-chain or cyclic alkyls, mono- orpolycyclic aryls, branched, straight-chain or cyclic heteroalkyls, mono-or polycyclic heteroaryls, halos, branched, straight-chain or cyclichaloalkyls, hydroxyls, oxos, thioxos, branched, straight-chain or cyclicalkoxys, branched, straight-chain or cyclic haloalkoxys,trifluoromethoxys, mono- or polycyclic aryloxys, mono- or polycyclicheteroaryloxys, ethers, alcohols, sulfides, thioethers, sulfanyls(thiols), imines, azos, azides, amines (primary, secondary andtertiary), nitriles (any isomer), cyanates (any isomer), thiocyanates(any isomer), nitrosos, nitros, diazos, sulfoxides, sulfonyls, sulfonicacids, sulfamides, sulfonamides, sulfamic esters, aldehydes, ketones,carboxylic acids, esters, amides, amidines, formadines, amino acids,acetylenes, carbamates, lactones, lactams, glucosides, gluconurides,sulfones, ketals, acetals, thioketals, oximes, oxamic acids, oxamicesters, etc., and combinations of these groups. Substituent groupsbearing reactive functionalities may be protected or unprotected, as iswell-known in the art.

In one illustrative embodiment, the spiro 2,4-pyrimidinediaminecompounds described herein are compounds according to structural formula(I):

including salts, hydrates, solvates and N-oxides thereof, wherein:

-   -   L¹ is a direct bond or a linker;    -   L² is a direct bond or a linker;    -   R² is selected from the group consisting of (C1-C6) alkyl        optionally substituted with one or more of the same or different        R⁸ groups, (C3-C8) alkyl optionally substituted with one or more        of the same or different R⁸ groups, 3-8 membered        cycloheteroalkyl optionally substituted with one or more of the        same or different R⁸ groups, (C5-C15) aryl optionally        substituted with one or more of the same or different R⁸ groups,        phenyl optionally substituted with one or more of the same or        different R⁸ groups and 5-15 membered heteroaryl optionally        substituted with one or more of the same or different R⁸ groups;    -   R⁴ is

-   -   each W is, independently of the other, —CR³¹R³¹—;    -   X is selected from the group consisting of —N— and —CH—;    -   Y and Z are each, independently of one another, selected from        the group consisting of —O—, —S—, —SO—, —SO₂—, —SONR³⁶—, —NH—,        —NR³⁵— and —NR³⁷—;    -   R⁵ is selected from the group consisting of hydrogen, an        electronegative group, —OR^(d), —SR^(d), (C1-C3) haloalkyloxy,        (C1-C3) perhaloalkyloxy, —NR^(c)R^(c), halogen, (C1-C3)        haloalkyl, (C1-C3) perhaloalkyl, —CF₃, —CH₂CF₃, —CF₂CF₃, —CN,        —NC, —OCN, —SCN, —NO, —NO₂, —N₃, —S(O)_(R) ^(d), —S(O)₂R^(d),        —S(O)₂OR^(d), —S(O)NR^(c)R^(c); —S(O)₂NR^(c)R^(c), —OS(O)R^(d),        —OS(O)₂R^(d), —OS(O)₂OR^(d), —OS(O)NR^(c)R^(c),        —OS(O)₂NR^(c)R^(c), —C(O)R^(d), —C(O)OR^(d), —C(O)NR^(c)R^(c),        —C(NH)NR^(c)R^(c), —OC(O)R^(d), —SC(O)R^(d), —OC(O)OR^(d),        —SC(O)OR^(d), —OC(O)NR^(c)R^(c), —SC(O)NR^(c)R^(c),        —OC(NH)NR^(c)R^(c), —SC(NH)NR^(c)R^(c), —[NHC(O)]_(n)R^(d),        [NHC(O)]_(n)OR^(d), —[NHC(O)]_(n)NR^(c)R^(c) and        —[NHC(NH)]_(n)NR^(c)R^(c), (C5-C10) aryl optionally substituted        with one or more of the same or different R⁸ groups, phenyl        optionally substituted with one or more of the same or different        R⁸ groups, (C6-C16) arylalkyl optionally substituted with one or        more of the same or different R⁸ groups, 5-10 membered        heteroaryl optionally substituted with one or more of the same        or different R⁸ groups and 6-16 membered heteroarylalkyl        optionally substituted with one or more of the same or different        R⁸ groups, (C1-C6) alkyl optionally substituted with one or more        of the same or different R⁸ groups, (C1-C4) alkanyl optionally        substituted with one or more of the same or different R⁸ groups,        (C2-C4) alkenyl optionally substituted with one or more of the        same or different R⁸ groups and (C2-C4) alkynyl optionally        substituted with one or more of the same or different R⁸ groups;    -   R⁶ independently is selected from the group consisting of        hydrogen, an electronegative group, —OR^(d), —SR^(d), (C1-C3)        haloalkyloxy, (C1-C3) perhaloalkyloxy, —NR^(c)R^(c), halogen,        (C1-C3) haloalkyl, (C1-C3) perhaloalkyl, —CF₃, —CH₂CF₃, —CF₂CF₃,        —CN, —NC, —OCN, —SCN, —NO, —NO₂, —N₃, —S(O)R^(d), —S(O)₂R^(d),        —S(O)₂OR^(d), —S(O)NR^(c)R^(c); —S(O)₂NR^(c)R^(c), —OS(O)R^(d),        —OS(O)₂R^(d), —OS(O)₂OR^(d), —OS(O)NR^(c)R^(c),        —OS(O)₂NR^(c)R^(c), —C(O)R^(d)—C(O)OR^(d), —C(O)NR^(c)R^(c),        —C(NH)NR^(c)R^(c), —OC(O)R^(d), —SC(O)R^(d), —OC(O)OR^(d),        —SC(O)OR^(d), —OC(O)NR^(c)R^(c), —SC(O)NR^(c)R^(c),        —OC(NH)NR^(c)R^(c), —SC(NH)NR^(c)R^(c), —[NHC(O)]_(n)R^(d),        —[NHC(O)]_(n)OR^(d), —[NHC(O)]_(n)NR^(c)R^(c) and        —[NHC(NH)]_(n)NR^(c)R^(c), (C5-C10) aryl optionally substituted        with one or more of the same or different R⁸ groups, phenyl        optionally substituted with one or more of the same or different        R⁸ groups, (C6-C16) arylalkyl optionally substituted with one or        more of the same or different R⁸ groups, 5-10 membered        heteroaryl optionally substituted with one or more of the same        or different R⁸ groups and 6-16 membered heteroarylalkyl        optionally substituted with one or more of the same or different        R⁸ groups;

R⁸ is selected from the group consisting of R^(a), R^(b), R^(a)substituted with one or more of the same or different R^(a) or R^(b),—OR^(a) substituted with one or more of the same or different R^(a) orR^(b), —B(OR^(a))₂, —B(NR^(c)R^(c))₂, —(CH₂)_(m)—R^(b),—(CHR^(a))_(m)—R^(b), —O—(CH₂)_(m)—R^(b), —S—(CH₂)_(m)—R^(b),—O—CHR^(a)R^(b), —O—CR^(a)(R^(b))₂, —O—(CHR^(a))_(m)—R^(b), —O—(CH₂)_(m)—CH[(CH₂)_(m)R^(b)]R^(b), —S—(CHR^(a))_(m)—R^(b),—C(O)NH—(CH₂)_(m)—R^(b), —C(O)NH—(CHR^(a))_(m)—R^(b),—O—(CH₂)_(m)—C(O)NH—(CH₂)_(m)—R^(b),—S—(CH₂)_(m)—C(O)NH—(CH₂)_(m)—R^(b),—O—(CHR^(a))_(m)—C(O)NH—(CHR^(a))_(m)—R^(b),—S—(CHR^(a))_(m)—C(O)NH—(CHR^(a))_(m)—R^(b), NH—(CH₂)_(m)—R^(b),—NH—(CHR^(a))_(m)—R^(b), —NH[(CH₂)_(m)R^(b)], —N[(CH₂)_(m)R^(b)]₂,—NHC(O)—NH—(CH₂)_(m)—R^(b), —NH—C(O)—(CH₂)_(m)—CHR^(b)R^(b) and—NH—(CH₂)_(m)—C(O)—NH—(CH₂)_(m)—R^(b);

-   -   each R³¹ is, independently of the others, hydrogen or (C1-C6)        alkyl optionally substituted with one or more of the same or        different R⁸ groups;    -   each R³⁵ is, independently of the other, selected from the group        consisting of hydrogen and R⁸, or, alternatively, the two R³⁵        groups are taken together to form an oxo (═O), or ═NR³⁸ group;    -   each R³⁶ is, independently of the others, selected from the        group consisting of hydrogen and (C1-C6) alkyl;    -   each R³⁷ is, independently of the others, selected from the        group consisting of hydrogen and a progroup;    -   R³⁸ is selected from the group consisting of hydrogen, (C1-C6)        alkyl and (C5-C14) aryl;    -   each R^(a) is, independently of the others, selected from the        group consisting of hydrogen, (C1-C6) alkyl, (C3-C8) cycloalkyl,        cyclohexyl, (C4-C11) cycloalkylalkyl, (C5-C10) aryl, phenyl,        (C6-C16) arylalkyl, benzyl, 2-6 membered heteroalkyl, 3-8        membered cycloheteroalkyl, morpholinyl, piperazinyl,        homopiperazinyl, piperidinyl, 4-11 membered        cycloheteroalkylalkyl, 5-10 membered heteroaryl and 6-16        membered heteroarylalkyl;

each R^(b) is, independently of the others, a suitable groupindependently selected from the group consisting of ═O, —OR^(d), (C1-C3)haloalkyloxy, —OCF₃, ═S, —SR^(d), ═NR^(d), ═NOR^(d), —NR^(c)R^(c),halogen, —CF₃, —CN, —NC, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)R^(d),—S(O)₂R^(d), —S(O)₂OR^(d), —S(O)NR^(c)R^(c), —S(O)₂NR^(c)R^(c),—OS(O)R^(d), —OS(O)₂R^(d), —OS(O)₂OR^(d), —OS(O)₂NR^(c)R^(c),—C(O)R^(d), —C(O)OR^(d), —C(O)NR^(c)R^(c), —C(NH)NR^(c)R^(c),—C(NR^(a))NR^(c)R^(c), —C(NOH)R^(a), —C(NOH)NR^(c)R^(c), —OC(O)R^(d),—OC(O)OR^(d), —OC(O)NR^(c)R^(c), —OC(NH)NR^(c)R^(c),—OC(NR^(a))NR^(c)R^(c), —[NHC(O)]_(n)R^(d), —[NR^(a)C(O)]_(n)R^(d),—[NHC(O)]_(n)OR^(d), —[NR^(a)C(O)]_(n)OR^(d), —[NHC(O)]_(n)NR^(c)R^(c),—[NR^(a)C(O)]_(n)NR^(c)R^(c), —[NHC(NH)]_(n)NR^(c)R^(c) and—[NR^(a)C(NR^(a))]_(n)NR^(c)R^(c);

-   -   each R^(c) is, independently or the others, a protecting group        or R^(a), or, alternatively, each R^(c) is taken together with        the nitrogen atom to which it is bonded to form a 5 to        8-membered cycloheteroalkyl or heteroaryl which may optionally        include one or more of the same or different additional        heteroatoms and which may optionally be substituted with one or        more of the same or different R^(a) or suitable R^(b) groups;    -   each R^(d) is, independently of the others, a protecting group        or R^(a);    -   each m is, independently of the others, an integer from 1 to 3;    -   each n is, independently of the others, an integer from 0 to 3;        and    -   o is an integer from 1 to 6.

In one embodiment, R⁵ is F and R⁶ is hydrogen.

In another aspect, prodrugs of the Spiro 2,4-pyrimidinediamine compoundsare provided. Such prodrugs may be active in their prodrug form, or maybe inactive until converted under physiological or other conditions ofuse to an active drug form. In the prodrugs described herein, one ormore functional groups of the spiro 2,4-pyrimidinediamine compounds areincluded in promoieties that cleave from the molecule under theconditions of use, typically by way of hydrolysis, enzymatic cleavage orsome other cleavage mechanism, to yield the functional groups. Forexample, primary or secondary amino groups may be included in an amidepromoiety that cleaves under conditions of use to generate the primaryor secondary amino group. Thus, the prodrugs described herein includespecial types of protecting groups, termed “progroups,” masking one ormore functional groups of the spiro 2,4-pyrimidinediamine compounds thatcleave under the conditions of use to yield an active spiro2,4-pyrimidinediamine drug compound. Functional groups within the2,4-pyrimidinediamine compounds that may be masked with progroups forinclusion in a promoiety include, but are not limited to, amines(primary and secondary), hydroxyls, sulfanyls (thiols), carboxyls,carbonyls, phenols, catechols, diols, alkynes, phosphates, etc. Myriadprogroups suitable for masking such functional groups to yieldpromoieties that are cleavable under the desired conditions of use areknown in the art. All of these progroups, alone or in combinations, maybe included in the prodrugs of the invention. Specific examples ofpromoieties that yield primary or secondary amine groups that can beincluded in the prodrugs of the invention include, but are not limitedto amides, carbamates, imines, ureas, phosphenyls, phosphoryls andsulfenyls. Specific examples of promoieties that yield sulfanyl groupsthat can be included in the prodrugs of the invention include, but arenot limited to, thioethers, for example S-methyl derivatives (monothio,dithio, oxythio, aminothio acetals), silyl thioethers, thioesters,thiocarbonates, thiocarbamates, asymmetrical disulfides, etc. Specificexamples of promoieties that cleave to yield hydroxyl groups that can beincluded in the prodrugs of the invention include, but are not limitedto, sulfonates, esters and carbonates. Specific examples of promoietiesthat yield carboxyl groups that can be included in the prodrugsdescribed herein included, but are not limited to, esters (includingsilyl esters, oxamic acid esters and thioesters), amides and hydrazides.

In one illustrative embodiment, prodrugs include compounds according tostructural formula (I) in which the protecting group of R^(c) and R^(d)is a progroup.

In another illustrative embodiment, prodrugs are compounds according tostructural formula (II):

including salts, hydrates, solvates and N-oxides thereof, wherein:

-   -   R², R⁴, R⁵, R⁶, L¹ and L² are as previously defined for        structural formula (I);    -   R^(2b) is a progroup;    -   R^(4b) is progroup or an alkyl group, e.g., methyl.

In another aspect, pharmaceutical compositions comprising one or morecompounds and/or prodrugs described herein and an appropriate carrier,excipient or diluent are provided. The exact nature of the carrier,excipient or diluent will depend upon the desired use for thecomposition, and may range from being suitable or acceptable forveterinary uses to being suitable or acceptable for human use.

In still another aspect, intermediates useful for synthesizing the2,4-pyrimidinediamine compounds and prodrugs described herein areprovided. In one embodiment, the intermediates are spiro compoundsaccording to structural formula (III):

including salts, hydrates, solvates and N-oxides thereof, wherein o, W.R³⁵, X, Y and Z are as previously defined for structural formula (I) andD is hydrogen, halogen, —NO₂ or —NH₂.

In another embodiment, the intermediates are 2-4-pyrimidineaminesaccording to structural formula (IV):

including salts, hydrates, solvates and N-oxides thereof, wherein R⁵,R⁶, o, R³¹, R³⁵, X, Y and Z are as previously defined for structuralformula (I) and LG is a leaving group, such as, for example, —S(O)₂Me,—SMe or halo (e.g., F, Cl, Br, J).

The spiro 2,4-pyrimidinediamine compounds described herein are potentinhibitors of degranulation of immune cells, such as mast, basophil,neutrophil and/or eosinophil cells. Thus, in still another aspect,methods of regulating, and in particular, inhibiting, degranulation ofsuch cells are provided. The method generally involves contacting a cellthat degranulates with an amount of a spiro 2,4-pyrimidinediaminecompound or prodrug thereof, or an acceptable salt, hydrate, solvate,N-oxide and/or composition thereof, effective to regulate or inhibitdegranulation of the cell. The method may be practiced in in vitrocontexts or in in vivo contexts as a therapeutic approach towards thetreatment or prevention of diseases characterized by, caused by orassociated with cellular degranulation.

While not intending to be bound by any theory of operation, spiro2,4-pyrimidinediamine compounds may exert their degranulation inhibitoryeffect, at least in part, by blocking or inhibiting the signaltransduction cascade(s) initiated by crosslinking of the high affinityFc receptors for IgE (“FcεRI”) and/or IgG (“FcγRI”). Indeed, the spiro2,4-pyrimidinediamine compounds may be inhibitors of both FcεRI-mediatedand FcγRI-mediated degranulation. As a consequence, the 2,4-pyrimidinecompounds may be used to inhibit these Fc receptor signaling cascades inany cell type expressing such FcεRI and/or FcγRI receptors including butnot limited to macrophages, mast, basophil, neutrophil and/or eosinophilcells.

The methods may also permit the regulation of, and in particular theinhibition of, downstream processes that result as a consequence ofactivating such Fc receptor signaling cascade(s). Such downstreamprocesses include, but are not limited to, FcεRI-mediated and/orFcγRI-mediated degranulation, cytokine production and/or the productionand/or release of lipid mediators such as leukotrienes andprostaglandins. The method generally involves contacting a cellexpressing an Fc receptor, such as one of the cell types discussedabove, with an amount of a spiro 2,4-pyrimidinediamine compound orprodrug thereof, or an acceptable salt, hydrate, solvent, N-oxide and/orcomposition thereof, effective to regulate or inhibit the Fc receptorsignaling cascade and/or a downstream process effected by the activationof this signaling cascade. The method may be practiced in in vitrocontexts or in in vivo contexts as a therapeutic approach towards thetreatment or prevention of diseases characterized by, caused by orassociated with the Fc receptor signaling cascade, such as diseaseseffected by the release of granule specific chemical mediators upondegranulation, the release and/or synthesis of cytokines and/or therelease and/or synthesis of lipid mediators such as leukotrienes andprostaglandins.

In yet another aspect, methods of treating and/or preventing diseasescharacterized by, caused by or associated with the release of chemicalmediators as a consequence of activating Fc receptor signaling cascades,such as FcεRI and/or FcγRI-signaling cascades is provided. The methodsmay be practiced in animals in veterinary contexts or in humans. Themethods generally involve administering to an animal subject or human anamount of a spiro 2,4-pyrimidinediamine compound or prodrug thereof, oran acceptable salt, hydrate, solvate, N-oxide and/or compositionthereof, effective to treat or prevent the disease. As discussedpreviously, activation of the FcεRI or FcγRI receptor signaling cascadein certain immune cells leads to the release and/or synthesis of avariety of chemical substances that are pharmacological mediators of awide variety of diseases. Any of these diseases may be treated orprevented according to the methods of the invention.

For example, in mast cells and basophil cells, activation of the FcεRIor FcγRI signaling cascade leads to the immediate (i.e., within 1-3 min.of receptor activation) release of preformed mediators of atopic and/orType I hypersensitivity reactions (e.g., histamine, proteases such astryptase, etc.) via the degranulation process. Such atopic or Type Ihypersensitivity reactions include, but are not limited to, anaphylacticreactions to environmental and other allergens (e.g., pollens, insectand/or animal venoms, foods, drugs, contrast dyes, etc.), anaphylactoidreactions, hay fever, allergic conjunctivitis, allergic rhinitis,allergic asthma, atopic dermatitis, eczema, urticaria, mucosaldisorders, tissue disorders and certain gastrointestinal disorders.

The immediate release of the preformed mediators via degranulation isfollowed by the release and/or synthesis of a variety of other chemicalmediators, including, among other things, platelet activating factor(PAF), prostaglandins and leukotrienes (e.g., LTC4) and the de novosynthesis and release of cytokines such as TNFα, IL-4, IL-5, IL-6,IL-13, etc. The first of these two processes occurs approximately 3-30min. following receptor activation; the latter approximately 30 min.-7hrs. following receptor activation. These “late stage” mediators arethought to be in part responsible for the chronic symptoms of theabove-listed atopic and Type I hypersensitivity reactions, and inaddition are chemical mediators of inflammation and inflammatorydiseases (e.g., osteoarthritis, inflammatory bowel disease, ulcerativecolitis, Crohn's disease, idiopathic inflammatory bowel disease,irritable bowel syndrome, spastic colon, etc.), low grade scarring(e.g., scleroderma, increased fibrosis, keloids, post-surgical scars,pulmonary fibrosis, vascular spasms, migraine, reperfusion injury andpost myocardial infarction), and sicca complex or syndrome. All of thesediseases may be treated or prevented according to the methods of theinvention.

Additional diseases which can be treated or prevented according to themethods described herein include diseases associated with basophil celland/or mast cell pathology. Examples of such diseases include, but arenot limited to, diseases of the skin such as scleroderma, cardiacdiseases such as post myocardial infarction, pulmonary diseases such aspulmonary muscle changes or remodeling and chronic obstructive pulmonarydisease (COPD) and diseases of the gut such as inflammatory bowelsyndrome (spastic colon).

Spiro 2,4-pyrimidinediamine compounds are also potent inhibitors of thetyrosine kinase Syk kinase. Thus, in still another aspect, methods ofregulating, and in particular inhibiting, Syk kinase activity areprovided. The method generally involves contacting a Syk kinase or acell comprising a Syk kinase with an amount of a spiro2,4-pyrimidinediamine compound or prodrug thereof, or an acceptablesalt, hydrate, solvate, N-oxide and/or composition thereof, effective toregulate or inhibit Syk kinase activity. In one embodiment, the Sykkinase is an isolated or recombinant Syk kinase. In another embodiment,the Syk kinase is an endogenous or recombinant Syk kinase expressed by acell, for example a mast cell or a basophil cell. The method may bepracticed in in vitro contexts or in in vivo contexts as a therapeuticapproach towards the treatment or prevention of diseases characterizedby, caused by or associated with Syk kinase activity.

While not intending to be bound by any particular theory of operation,spiro 2,4-pyrimdinediamine compounds may inhibit cellular degranulationand/or the release of other chemical mediators primarily by inhibitingSyk kinase that gets activated through the gamma chain homodimer ofFcεRI (see, e.g., FIG. 2). This gamma chain homodimer is shared by otherFc receptors, including FcγRI, FcγRIII and FcαRI. For all of thesereceptors, intracellular signal transduction is mediated by the commongamma chain homodimer. Binding and aggregation of those receptorsresults in the recruitment and activation of tyrosine kinases such asSyk kinase. As a consequence of these common signaling activities, thespiro 2,4-pyrimidinediamine compounds described herein may be used toregulate, and in particular inhibit, the signaling cascades of Fcreceptors having this gamma chain homodimer, such as FcεRI, FcγRI,FcγRIII and FcαRI, as well as the cellular responses elicited throughthese receptors.

Syk kinase is known to play a critical role in other signaling cascades.For example, Syk kinase is an effector of B-cell receptor (BCR)signaling (Turner et al., 2000, Immunology Today 21:148-154) and is anessential component of integrin beta(1), beta(2) and beta(3) signalingin neutrophils (Mocsai et al., 2002, Immunity 16:547-558). As the spiro2,4-pyrimidinediamine compounds described herein are potent inhibitorsof Syk kinase, they can be used to regulate, and in particular inhibit,any signaling cascade where Syk plays a role, such as, fore example, theFc receptor, BCR and integrin signaling cascades, as well as thecellular responses elicited through these signaling cascades. Theparticular cellular response regulated or inhibited will depend, inpart, on the specific cell type and receptor signaling cascade, as iswell known in the art. Non-limiting examples of cellular responses thatmay be regulated or inhibited with the spiro 2,4-pyrimidinediaminecompounds include a respiratory burst, cellular adhesion, cellulardegranulation, cell spreading, cell migration, phagocytosis (e.g., inmacrophages), calcium ion flux (e.g., in mast, basophil, neutrophil,eosinophil and B-cells), platelet aggregation, and cell maturation(e.g., in B-cells).

Thus, in another aspect, methods of regulating, and in particularinhibiting, signal transduction cascades in which Syk plays a role areprovided. The methods generally involve contacting a Syk-dependentreceptor or a cell expressing a Syk-dependent receptor with an amount ofa spiro 2,4-pyrimidinediamine compound or prodrug described herein, oran acceptable salt, hydrate, solvate, N-oxide and/or compositionthereof, effective to regulate or inhibit the signal transductioncascade. The methods may also be used to regulate, and in particularinhibit, downstream processes or cellular responses elicited byactivation of the particular Syk-dependent signal transduction cascade.The methods may be practiced to regulate any signal transduction cascadewhere Syk is not known or later discovered to play a role. The methodsmay be practiced in in vitro contexts or in in vivo contexts as atherapeutic approach towards the treatment or prevention of diseasescharacterized by, caused by or associated with activation of theSyk-dependent signal transduction cascade. Non-limited examples of suchdiseases include those previously discussed.

Spiro 2,4-pyrimidinediamine compounds described herein may also be usedto treat or prevent autoimmune diseases and/or symptoms of suchdiseases. The methods generally involve administering to a subjectsuffering from an autoimmune disease or at risk of developing anautoimmune disease an amount of a spiro 2,4-pyrimidinediamine method orprodrug thereof, or an acceptable salt, N-oxide, hydrate, solvate orcomposition thereof, effective to treat or prevent the autoimmunedisease and/or its associated symptoms. Autoimmune diseases that may betreated or prevented with the spiro 2,4-pyrimidinediamine compoundsinclude those diseases that are commonly associated with nonanaphylactichypersensitivity reactions (Type II, Type III and/or Type IVhypersensitivity reactions) and/or those diseases that are mediated, atleast in part, by activation of the FcγR signaling cascade in monocytecells. Such autoimmune disease include, but are not limited to, thoseautoimmune diseases that are frequently designated as single organ orsingle cell-type autoimmune disorders and those autoimmune disease thatare frequently designated as involving systemic autoimmune disorder.Non-limiting examples of diseases frequently designated as single organor single cell-type autoimmune disorders include: Hashimoto'sthyroiditis, autoimmune hemolytic anemia, autoimmune atrophic gastritisof pernicious anemia, autoimmune encephalomyelitis, autoimmune orchitis,Goodpasture's disease, autoimmune thrombocytopenia, sympatheticophthalmia, myasthenia gravis, Graves' disease, primary biliarycirrhosis, chronic aggressive hepatitis, ulcerative colitis andmembranous glomerulopathy. Non-limiting examples of diseases oftendesignated as involving systemic autoimmune disorder include: systemiclupus erythematosis, rheumatoid arthritis, Sjogren's syndrome, Reiter'ssyndrome, polymyositis-dermatomyositis, systemic sclerosis,polyarteritis nodosa, multiple sclerosis and bullous pemphigoid.

5. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a cartoon illustrating allergen-induced production ofIgE and consequent release of preformed and other chemical mediatorsfrom mast cells;

FIG. 2 provides a cartoon illustrating the FcεRI signal transductioncascade leading to degranulation of mast and/or basophil cells; and

FIG. 3 provides a cartoon illustrating the putative points of action ofcompounds that selectively inhibit upstream FcεRI-mediated degranulationand compounds that inhibit both FcεRI-mediated and ionomycin-induceddegranulation.

6. DETAILED DESCRIPTION 6.2 Definitions

As used herein, the following terms are intended to have the followingmeanings:

“Alkyl” by itself or as part of another substituent refers to asaturated or unsaturated branched, straight-chain or cyclic monovalenthydrocarbon radical having the stated number of carbon atoms (i.e.,C1-C6 means one to six carbon atoms) that is derived by the removal ofone hydrogen atom from a single carbon atom of a parent alkane, alkeneor alkyne. Typical alkyl groups include, but are not limited to, methyl;ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-1-yl,propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl,prop-2-en-1-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl,prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls such as butan-1-yl,butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl,cyclobutane-1-yl, but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl,but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl,but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. Wherespecific levels of saturation are intended, the nomenclature “alkanyl,”“alkenyl” and/or “alkynyl” is used, as defined below. In someembodiments, the alkanyl groups are (C₁-C₁₀) alkyl. In otherembodiments, the alkyl groups are (C₁-C₆) alkyl.

“Alkanyl” by itself or as part of another substituent refers to asaturated branched, straight-chain or cyclic alkyl derived by theremoval of one hydrogen atom from a single carbon atom of a parentalkane. Typical alkanyl groups include, but are not limited to,methanyl; ethanyl; propanyls such as propan-1-yl, propan-2-yl(isopropyl), cyclopropan-1-yl, etc.; butanyls such as butan-1-yl,butan-2-yl (sec-butyl), 2-methyl-propan-1-yl (isobutyl),2-methyl-propan-2-yl (t-butyl), cyclobutane-1-yl, etc.; and the like.

“Alkenyl” by itself or as part of another substituent refers to anunsaturated branched, straight-chain or cyclic alkyl having at least onecarbon-carbon double bond derived by the removal of one hydrogen atomfrom a single carbon atom of a parent alkene. The group may be in eitherthe cis or trans conformation about the double bond(s). Typical alkenylgroups include, but are not limited to, ethenyl; propenyls such asprop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, prop-2-en-2-yl,cycloprop-1-en-1-yl; cycloprop-2-en-1-yl; butenyls such asbut-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, etc.;and the like. In preferred embodiments, the alkenyl group is (C2-C6)alkenyl.

“Alkynyl” by itself or as part of another substituent refers to anunsaturated branched, straight-chain or cyclic alkyl having at least onecarbon-carbon triple bond derived by the removal of one hydrogen atomfrom a single carbon atom of a parent alkyne. Typical alkynyl groupsinclude, but are not limited to, ethynyl; propynyls such asprop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl,but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like.

“Alkyldiyl” by itself or as part of another substituent refers to asaturated or unsaturated, branched, straight-chain or cyclic divalenthydrocarbon group having the stated number of carbon atoms (i.e., C₁-C₆means from one to six carbon atoms) derived by the removal of onehydrogen atom from each of two different carbon atoms of a parentalkane, alkene or alkyne, or by the removal of two hydrogen atoms from asingle carbon atom of a parent alkane, alkene or alkyne. The twomonovalent radical centers or each valency of the divalent radicalcenter can form bonds with the same or different atoms. Typicalalkyldiyl groups include, but are not limited to, methandiyl; ethyldiylssuch as ethan-1,1-diyl, ethan-1,2-diyl, ethen-1,1-diyl, ethen-1,2-diyl;propyldiyls such as propan-1,1-diyl, propan-1,2-diyl, propan-2,2-diyl,propan-1,3-diyl, cyclopropan-1,1-diyl, cyclopropan-1,2-diyl,prop-1-en-1,1-diyl, prop-1-en-1,2-diyl, prop-2-en-1,2-diyl,prop-1-en-1,3-diyl, cycloprop-1-en-1,2-diyl, cycloprop-2-en-1,2-diyl,cycloprop-2-en-1,1-diyl, prop-1-yn-1,3-diyl, etc.; butyldiyls such as,butan-1,1-diyl, butan-1,2-diyl, butan-1,3-diyl, butan-1,4-diyl,butan-2,2-diyl, 2-methyl-propan-1,1-diyl, 2-methyl-propan-1,2-diyl,cyclobutane-1,1-diyl; cyclobutane-1,2-diyl, cyclobutane-1,3-diyl,but-1-en-1,1-diyl, but-1-en-1,2-diyl, but-1-en-1,3-diyl,but-1-en-1,4-diyl, 2-methyl-prop-1-en-1,1-diyl,2-methanylidene-propan-1,1-diyl, buta-1,3-dien-1,1-diyl,buta-1,3-dien-1,2-diyl, buta-1,3-dien-1,3-diyl, buta-1,3-dien-1,4-diyl,cyclobut-1-en-1,2-diyl, cyclobut-1-en-1,3-diyl, cyclobut-2-en-1,2-diyl,cyclobuta-1,3-dien-1,2-diyl, cyclobuta-1,3-dien-1,3-diyl,but-1-yn-1,3-diyl, but-1-yn-1,4-diyl, buta-1,3-diyn-1,4-diyl, etc.; andthe like. Where specific levels of saturation are intended, thenomenclature alkanyldiyl, alkenyldiyl and/or alkynyldiyl is used. Whereit is specifically intended that the two valencies are on the samecarbon atom, the nomenclature “alkylidene” is used. In some embodiments,the alkyldiyl groups are (C₁-C₁₀) alkyldiyl. In other embodiments, thealkyldiyl groups are (C₁-C₆) alkyldiyl. Also preferred are saturatedacyclic alkanyldiyl groups in which the radical centers are at theterminal carbons, e.g., methandiyl (methano); ethan-1,2-diyl (ethano);propan-1,3-diyl (propano); butan-1,4-diyl (butano); and the like (alsoreferred to as alkylenos, defined infra).

“Alkyleno” by itself or as part of another substituent refers to astraight-chain saturated or unsaturated alkyldiyl group having twoterminal monovalent radical centers derived by the removal of onehydrogen atom from each of the two terminal carbon atoms ofstraight-chain parent alkane, alkene or alkyne. The locant of a doublebond or triple bond, if present, in a particular alkyleno is indicatedin square brackets. Typical alkyleno groups include, but are not limitedto, methano; ethylenos such as ethano, etheno, ethyno; propylenos suchas propano, prop[1]eno, propa[1,2]dieno, prop[1]yno, etc.; butylenossuch as butano, but[1]eno, but[2]eno, buta[1,3]dieno, but[1]yno,but[2]yno, buta[1,3]diyno, etc.; and the like. Where specific levels ofsaturation are intended, the nomenclature alkano, alkeno and/or alkynois used. In some embodiments, the alkyleno group is (C₁-C₁₀) alkyleno.In other embodiments, the alkyleno group is (C₁-C₆) alkyleno. In stillother embodiments, the alkyleno group is (C₁-C₃) alkyleno. Alsopreferred are straight-chain saturated alkano groups, e.g., methanolethano, propano, butano, and the like.

“Heteroalkyl,” Heteroalkanyl,” Heteroalkenyl,” Heteroalkynyl,”Heteroalkyldiyl” and “Heteroalkyleno” by themselves or as part ofanother substituent refer to alkyl, alkanyl, alkenyl, alkynyl, alkyldiyland alkyleno groups, respectively, in which one or more of the carbonatoms are each independently replaced with the same or differentheteroatoms or heteroatomic groups. Typical heteroatoms and/orheteroatomic groups which can replace the carbon atoms include, but arenot limited to, —O—, —S—, —S—O—, —NR′—, —PH—, —S(O)—, —S(O)₂—,—S(O)NR′—, —S(O)₂NR′—, and the like, including combinations thereof,where each R′ is independently hydrogen or (C₁-C₆) alkyl.

“Cycloalkyl” and “Heterocycloalkyl” by themselves or as part of anothersubstituent refer to cyclic versions of “alkyl” and “heteroalkyl”groups, respectively. For heteroalkyl groups, a heteroatom can occupythe position that is attached to the remainder of the molecule. Typicalcycloalkyl groups include, but are not limited to, cyclopropyl;cyclobutyls such as cyclobutanyl and cyclobutenyl; cyclopentyls such ascyclopentanyl and cyclopentenyl; cyclohexyls such as cyclohexanyl andcyclohexenyl; and the like. Typical heterocycloalkyl groups include, butare not limited to, tetrahydrofuranyl (e.g., tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, etc.), piperidinyl (e.g., piperidin-1-yl,piperidin-2-yl, etc.), morpholinyl (e.g., morpholin-3-yl,morpholin-4-yl, etc.), piperazinyl (e.g., piperazin-1-yl,piperazin-2-yl, etc.), and the like.

“Acyclic Heteroatomic Bridge” refers to a divalent bridge in which thebackbone atoms are exclusively heteroatoms and/or heteroatomic groups.Typical acyclic heteroatomic bridges include, but are not limited to,—O—, —S—, —S—O—, —NR′—, —PH—, —S(O)—, —S(O)₂—, —S(O)NR′—, —S(O)₂NR′—,and the like, including combinations thereof, where each R′ isindependently hydrogen or (C₁-C₆) alkyl.

“Parent Aromatic Ring System” refers to an unsaturated cyclic orpolycyclic ring system having a conjugated π electron system.Specifically included within the definition of “parent aromatic ringsystem” are fused ring systems in which one or more of the rings arearomatic and one or more of the rings are saturated or unsaturated, suchas, for example, fluorene, indane, indene, phenalene,tetrahydronaphthalene, etc. Typical parent aromatic ring systemsinclude, but are not limited to, aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexylene, indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, tetrahydronaphthalene, triphenylene, trinaphthalene, and thelike, as well as the various hydro isomers thereof.

“Aryl” by itself or as part of another substituent refers to amonovalent aromatic hydrocarbon group having the stated number of carbonatoms (i.e., C₅-C₁₅ means from 5 to 15 carbon atoms) derived by theremoval of one hydrogen atom from a single carbon atom of a parentaromatic ring system. Typical aryl groups include, but are not limitedto, groups derived from aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexylene, as-indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, triphenylene, trinaphthalene, and the like, as well as thevarious hydro isomers thereof. In some embodiments, the aryl group is(C₅-C₁₅) aryl, with (C₅-C₁₀) being even more preferred. Preferred arylsare cyclopentadienyl, phenyl and naphthyl.

“Arylaryl” by itself or as part of another substituent refers to amonovalent hydrocarbon group derived by the removal of one hydrogen atomfrom a single carbon atom of a ring system in which two or moreidentical or non-identical parent aromatic ring systems are joineddirectly together by a single bond, where the number of such direct ringjunctions is one less than the number of parent aromatic ring systemsinvolved. Typical arylaryl groups include, but are not limited to,biphenyl, triphenyl, phenyl-naphthyl, binaphthyl, biphenyl-naphthyl, andthe like. Where the number of carbon atoms in an arylaryl group arespecified, the numbers refer to the carbon atoms comprising each parentaromatic ring. For example, (C₅-C₁₅) arylaryl is an arylaryl group inwhich each aromatic ring comprises from 5 to 15 carbons, e.g., biphenyl,triphenyl, binaphthyl, phenylnaphthyl, etc. Preferably, each parentaromatic ring system of an arylaryl group is independently a (C₅-C₁₅)aromatic, more preferably a (C₅-C₁₀) aromatic. Also preferred arearylaryl groups in which all of the parent aromatic ring systems areidentical, e.g., biphenyl, triphenyl, binaphthyl, trinaphthyl, etc.

“Biaryl” by itself or as part of another substituent refers to anarylaryl group having two identical parent aromatic systems joineddirectly together by a single bond. Typical biaryl groups include, butare not limited to, biphenyl, binaphthyl, bianthracyl, and the like.Preferably, the aromatic ring systems are (C₅-C₁₅) aromatic rings, morepreferably (C₅-C₁₀) aromatic rings. A particularly preferred biarylgroup is biphenyl.

“Arylalkyl” by itself or as part of another substituent refers to anacyclic alkyl group in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or sp³ carbon atom, is replaced withan aryl group. Typical arylalkyl groups include, but are not limited to,benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl,2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl,2-naphthophenylethan-1-yl and the like. Where specific alkyl moietiesare intended, the nomenclature arylalkanyl, arylalkenyl and/orarylalkynyl is used. In preferred embodiments, the arylalkyl group is(C₆-C₂₁) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of thearylalkyl group is (C₁-C₆) and the aryl moiety is (C₅-C₁₅). In someembodiments the arylalkyl group is (C₆-C₁₃), e.g., the alkanyl, alkenylor alkynyl moiety of the arylalkyl group is (C₁-C₃) and the aryl moietyis (C₅-C₁₀).

“Parent Heteroaromatic Ring System” refers to a parent aromatic ringsystem in which one or more carbon atoms are each independently replacedwith the same or different heteroatoms or heteroatomic groups. Typicalheteroatoms or heteroatomic groups to replace the carbon atoms include,but are not limited to, N, NH, P, O, S, S(O), S(O)₂, Si, etc.Specifically included within the definition of “parent heteroaromaticring systems” are fused ring systems in which one or more of the ringsare aromatic and one or more of the rings are saturated or unsaturated,such as, for example, benzodioxan, benzofuran, chromane, chromene,indole, indoline, xanthene, etc. Also included in the definition of“parent heteroaromatic ring system” are those recognized rings thatinclude common substituents, such as, for example, benzopyrone and1-methyl-1,2,3,4-tetrazole. Specifically excluded from the definition of“parent heteroaromatic ring system” are benzene rings fused to cyclicpolyalkylene glycols such as cyclic polyethylene glycols. Typical parentheteroaromatic ring systems include, but are not limited to, acridine,benzimidazole, benzisoxazole, benzodioxan, benzodioxole, benzo furan,benzopyrone, benzothiadiazole, benzothiazole, benzotriazole,benzoxazine, benzoxazole, benzoxazoline, carbazole, β-carboline,chromane, chromene, cinnoline, furan, imidazole, indazole, indole,indoline, indolizine, isobenzofuran, isochromene, isoindole,isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine,oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline,phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole,pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline,quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole,thiophene, triazole, xanthene, and the like.

“Heteroaryl” by itself or as part of another substituent refers to amonovalent heteroaromatic group having the stated number of ring atoms(e.g., “5-14 membered” means from 5 to 14 ring atoms) derived by theremoval of one hydrogen atom from a single atom of a parentheteroaromatic ring system. Typical heteroaryl groups include, but arenot limited to, groups derived from acridine, benzimidazole,benzisoxazole, benzodioxan, benzodioxole, benzofuran, benzopyrone,benzothiadiazole, benzothiazole, benzotriazole, benzoxazine,benzoxazole, benzoxazoline, carbazole, β-carboline, chromane, chromene,cinnoline, furan, imidazole, indazole, indole, indoline, indolizine,isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline,isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and thelike, as well as the various hydro isomers thereof. In some embodiments,the heteroaryl group is a 5-14 membered heteroaryl, with 5-10 memberedheteroaryl being particularly preferred.

“Heteroaryl-Heteroaryl” by itself or as part of another substituentrefers to a monovalent heteroaromatic group derived by the removal ofone hydrogen atom from a single atom of a ring system in which two ormore identical or non-identical parent heteroaromatic ring systems arejoined directly together by a single bond, where the number of suchdirect ring junctions is one less than the number of parentheteroaromatic ring systems involved. Typical heteroaryl-heteroarylgroups include, but are not limited to, bipyridyl, tripyridyl,pyridylpurinyl, bipurinyl, etc. Where the number of atoms are specified,the numbers refer to the number of atoms comprising each parentheteroaromatic ring systems. For example, 5-15 memberedheteroaryl-heteroaryl is a heteroaryl-heteroaryl group in which eachparent heteroaromatic ring system comprises from 5 to 15 atoms, e.g.,bipyridyl, tripuridyl, etc. Preferably, each parent heteroaromatic ringsystem is independently a 5-15 membered heteroaromatic, more preferablya 5-10 membered heteroaromatic. Also preferred are heteroaryl-heteroarylgroups in which all of the parent heteroaromatic ring systems areidentical.

“Biheteroaryl” by itself or as part of another substituent refers to aheteroaryl-heteroaryl group having two identical parent heteroaromaticring systems joined directly together by a single bond. Typicalbiheteroaryl groups include, but are not limited to, bipyridyl,bipurinyl, biquinolinyl, and the like. Preferably, the heteroaromaticring systems are 5-15 membered heteroaromatic rings, more preferably5-10 membered heteroaromatic rings.

“Heteroarylalkyl” by itself or as part of another substituent refers toan acyclic alkyl group in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or sp³ carbon atom, is replaced with aheteroaryl group. Where specific alkyl moieties are intended, thenomenclature heteroarylalkanyl, heteroarylalkenyl and/orheteroarylalkynyl is used. In preferred embodiments, the heteroarylalkylgroup is a 6-21 membered heteroarylalkyl, e.g., the alkanyl, alkenyl oralkynyl moiety of the heteroarylalkyl is (C₁-C₆) alkyl and theheteroaryl moiety is a 5-15-membered heteroaryl. In particularlypreferred embodiments, the heteroarylalkyl is a 6-13 memberedheteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety is (C₁-C₃)alkyl and the heteroaryl moiety is a 5-10 membered heteroaryl.

“Halogen” or “Halo” by themselves or as part of another substituent,unless otherwise stated, refer to fluoro, chloro, bromo and iodo.

“Haloalkyl” by itself or as part of another substituent refers to analkyl group in which one or more of the hydrogen atoms is replaced witha halogen. Thus, the term “haloalkyl” is meant to includemonohaloalkyls, dihaloalkyls, trihaloalkyls, etc. up to perhaloalkyls.For example, the expression “(C₁-C₂) haloalkyl” includes fluoromethyl,difluoromethyl, trifluoromethyl, 1-fluoroethyl, 1,1-difluoroethyl,1,2-difluoroethyl, 1,1,1-trifluoroethyl, perfluoroethyl, etc.

The above-defined groups may include prefixes and/or suffixes that arecommonly used in the art to create additional well-recognizedsubstituent groups. As examples, “alkyloxy” or “alkoxy” refers to agroup of the formula —OR″, “alkylamine” refers to a group of the formula—NHR″ and “dialkylamine” refers to a group of the formula —NR″R″, whereeach R″ is independently an alkyl. As another example, “haloalkoxy” or“haloalkyloxy” refers to a group of the formula —OR′″, where R′″ is ahaloalkyl.

“Protecting group” refers to a group of atoms that, when attached to areactive functional group in a molecule, mask, reduce or prevent thereactivity of the functional group. Typically, a protecting group may beselectively removed as desired during the course of a synthesis.Examples of protecting groups can be found in Greene and Wuts,Protective Groups in Organic Chemistry, 3^(rd) Ed., 1999, John Wiley &Sons, NY and Harrison et al., Compendium of Synthetic Organic Methods,Vols. 1-8, 1971-1996, John Wiley & Sons, NY. Representative aminoprotecting groups include, but are not limited to, formyl, acetyl,trifluoroacetyl, benzyl, benzyloxycarbonyl (“CBZ”), tert-butoxycarbonyl(“Boc”), trimethylsilyl (“TMS”), 2-trimethylsilyl-ethanesulfonyl(“TES”), trityl and substituted trityl groups, allyloxycarbonyl,9-fluorenylmethyloxycarbonyl (“FMOC”), nitro-veratryloxycarbonyl(“NVOC”) and the like. Representative hydroxyl protecting groupsinclude, but are not limited to, those where the hydroxyl group iseither acylated or alkylated such as benzyl and trityl ethers, as wellas alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers (e.g.,TMS or TIPPS groups) and allyl ethers.

“Prodrug” refers to a derivative of an active spiro2,4-pyrimidinediamine compound (drug) that requires a transformationunder the conditions of use, such as within the body, to release theactive spiro 2,4-pyrimidinediamine drug. Prodrugs are frequently, butnot necessarily, pharmacologically inactive until converted into theactive drug. Prodrugs are typically obtained by masking a functionalgroup in the Spiro 2,4-pyrimidinediamine drug believed to be in partrequired for activity with a progroup (defined below) to form apromoiety which undergoes a transformation, such as cleavage, under thespecified conditions of use to release the functional group, and hencethe active spiro 2,4-pyrimidinediamine drug. The cleavage of thepromoiety may proceed spontaneously, such as by way of a hydrolysisreaction, or it may be catalyzed or induced by another agent, such as byan enzyme, by light, by acid or base, or by a change of or exposure to aphysical or environmental parameter, such as a change of temperature.The agent may be endogenous to the conditions of use, such as an enzymepresent in the cells to which the prodrug is administered or the acidicconditions of the stomach, or it may be supplied exogenously.

A wide variety of progroups, as well as the resultant promoieties,suitable for masking functional groups in the active2,4-pyrimidinediamines compounds to yield prodrugs are well-known in theart. For example, a hydroxyl functional group may be masked as asulfonate, ester or carbonate promoiety, which may be hydrolyzed in vivoto provide the hydroxyl group. An amino functional group may be maskedas an amide, carbamate, imine, urea, phosphenyl, phosphoryl or sulfenylpromoiety, which may be hydrolyzed in vivo to provide the amino group. Acarboxyl group may be masked as an ester (including silyl esters andthioesters), amide or hydrazide promoiety, which may be hydrolyzed invivo to provide the carboxyl group. Nitrogen protecting groups andnitrogen pro-drugs of the invention may include lower alkyl groups aswell as amides, carbamates, etc. Other specific examples of suitableprogroups and their respective promoieties will be apparent to those ofskill in the art.

“Progroup” refers to a type of protecting group that, when used to maska functional group within an active spiro 2,4-pyrimidinediamine drug toform a promoiety, converts the drug into a prodrug. Progroups aretypically attached to the functional group of the drug via bonds thatare cleavable under specified conditions of use. Thus, a progroup isthat portion of a promoiety that cleaves to release the functional groupunder the specified conditions of use. As a specific example, an amidepromoiety of the formula —NH—C(O)CH₃ comprises the progroup —C(O)CH₃.

“Fc Receptor” refers to a member of the family of cell surface moleculesthat binds the Fc portion (containing the specific constant region) ofan immunoglobulin. Each Fc receptor binds immunoglobulins of a specifictype. For example the Fcα receptor (“FcαR”) binds IgA, the FcεR bindsIgE and the FcγR binds IgG.

The FcαR family includes the polymeric Ig receptor involved inepithelial transport of IgA/IgM, the mycloid specific receptor RcαRI(also called CD89), the Fcα/μR and at least two alternative IgAreceptors (for a recent review see Monteiro & van de Winkel, 2003, Annu.Rev. Immunol, advanced e-publication. The FcαRI is expressed onneutrophils, eosinophils, monocytes/macrophages, dendritic cells andkupfer cells. The FcαRI includes one alpha chain and the FcR gammahomodimer that bears an activation motif (ITAM) in the cytoplasmicdomain and phosphorylates Syk kinase.

The FcεR family includes two types, designated FcεRI and FcεRII (alsoknown as CD23). FcεRI is a high affinity receptor (binds IgE with anaffinity of about 10¹⁰M⁻¹) found on mast, basophil and eosinophil cellsthat anchors monomeric IgE to the cell surface. The FcεRI possesses onealpha chain, one beta chain and the gamma chain homodimer discussedabove. The FcεRII is a low affinity receptor expressed on mononuclearphagocytes, B lymphocytes, eosinophils and platelets. The FcεRIIcomprises a single polypeptide chain and does not include the gammachain homodimer.

The FcγR family includes three types, designated FcγRI (also known asCD64), FcγRII (also known as CD32) and FcγRIII (also known as CD16).FcγRI is a high affinity receptor (binds IgG1 with an affinity of10⁸M⁻¹) found on mast, basophil, mononuclear, neutrophil, eosinophil,dendritic and phagocyte cells that anchors monomeric IgG to the cellsurface. The FcγRI includes one alpha chain and the gamma chain dimershared by FcαRI and FcεRI.

The FcγRII is a low affinity receptor expressed on neutrophils,monocytes, eosinophils, platelets and B lymphocytes. The FcγRII includesone alpha chain, and does not include the gamma chain homodimerdiscussed above.

The FcγRIII is a low affinity (binds IgG1 with an affinity of 5×10⁵M⁻¹)expressed on NK, eosinophil, macrophage, neutrophil and mast cells. Itcomprises one alpha chain and the gamma homodimer shared by FcαRI, FcεRIand FcγRI.

Skilled artisans will recognize that the subunit structure and bindingproperties of these various Fc receptors, cell types expressing them,are not completely characterized. The above discussion merely reflectsthe current state-of-the-art regarding these receptors (see, e.g.,Immunobiology: The Immune System in Health & Disease, 5^(th) Edition,Janeway et al., Eds, 2001, ISBN 0-8153-3642-x, Figure 9.30 at pp. 371),and is not intended to be limiting with respect to the myriad receptorsignaling cascades that can be regulated with the compounds describedherein.

“Fc Receptor-Mediated Deuranulation” or “Fc Receptor-InducedDeuranulation” refers to degranulation that proceeds via an Fc receptorsignal transduction cascade initiated by crosslinking of an Fc receptor.

“IgE-Induced Degranulation” or “FcεRI-Mediated Degranulation” refers todegranulation that proceeds via the IgE receptor signal transductioncascade initiated by crosslinking of FcεRI-bound IgE. The crosslinkingmay be induced by an IgE-specific allergen or other multivalent bindingagent, such as an anti-IgE antibody. Referring to FIG. 2, in mast and/orbasophil cells, the FcεRI signaling cascade leading to degranulation maybe broken into two stages: upstream and downstream. The upstream stageincludes all of the processes that occur prior to calcium ionmobilization (illustrated as “Ca²⁺” in FIG. 2; see also FIG. 3). Thedownstream stage includes calcium ion mobilization and all processesdownstream thereof. Compounds that inhibit FcεRI-mediated degranulationmay act at any point along the FcεRI-mediated signal transductioncascade. Compounds that selectively inhibit upstream FcεRI-mediateddegranulation act to inhibit that portion of the FcεRI signaling cascadeupstream of the point at which calcium ion mobilization is induced. Incell-based assays, compounds that selectively inhibit upstreamFcεRI-mediated degranulation inhibit degranulation of cells such as mastor basophil cells that are activated or stimulated with an IgE-specificallergen or binding agent (such as an anti-IgE antibody) but do notappreciably inhibit degranulation of cells that are activated orstimulated with degranulating agents that bypass the FcεRI signalingpathway, such as, for example the calcium ionophores ionomycin andA23187.

“IgG-Induced Degranulation” or “FcγRI-Mediated Degranulation” refers todegranulation that proceeds via the FcγRI signal transduction cascadeinitiated by crosslinking of FcγRI-bound IgG. The crosslinking may beinduced by an IgG-specific allergen or another multivalent bindingagent, such as an anti-IgG or fragment antibody. Like the FcεRIsignaling cascade, in mast and basophil cells the FcγRI signalingcascade also leads to degranulation which may be broken into the sametwo stages: upstream and downstream. Similar to FcεRI-mediateddegranulation, compounds that selectively inhibit upstreamFcγRI-mediated degranulation act upstream of the point at which calciumion mobilization is induced. In cell-based assays, compounds thatselectively inhibit upstream FcγRI-mediated degranulation inhibitdegranulation of cells such as mast or basophil cells that are activatedor stimulated with an IgG-specific allergen or binding agent (such as ananti-IgG antibody or fragment) but do not appreciably inhibitdegranulation of cells that are activated or stimulated withdegranulating agents that bypass the FcγRI signaling pathway, such as,for example the calcium ionophores ionomycin and A23187.

“Ionophore-Induced Degranulation” or “Ionophore-Mediated Degranulation”refers to degranulation of a cell, such as a mast or basophil cell, thatoccurs upon exposure to a calcium ionophore such as, for example,ionomycin or A23187.

“Syk Kinases” refers to the well-known 72 kDa non-receptor (cytoplasmic)spleen protein tyrosine kinase expressed in B-cells and otherhematopoetic cells. Syk kinase includes two consensus Src-homology 2(SH2) domains in tandem that bind to phosphorylated immunoreceptortyrosine-based activation motifs (“ITAMs”), a “linker” domain and acatalytic domain (for a review of the structure and function of Sykkinase see Sada et al., 2001, J. Biochem. (Tokyo) 130:177-186); see alsoTurner et al., 2000, Immunology Today 21:148-154). Syk kinase has beenextensively studied as an effector of B-cell receptor (BCR) signaling(Turner et al., 2000, supra). Syk kinase is also critical for tyrosinephosphorylation of multiple proteins which regulate important pathwaysleading from immunoreceptors, such as Ca²⁺ mobilization andmitogen-activated protein kinase (MAPK) cascades (see, e.g., FIG. 2) anddegranulation. Syk kinase also plays a critical role in integrinsignaling in neutrophils (see, e.g., Mocsai et al., 2002, Immunity16:547-558).

As used herein, Syk kinase includes kinases from any species of animal,including but not limited to, homosapiens, simian, bovine, porcine,rodent, etc., recognized as belonging to the Syk family. Specificallyincluded are isoforms, splice variants, allelic variants, mutants, bothnaturally occurring and man-made. The amino acid sequences of such Sykkinases are well known and available from GENBANK. Specific examples ofmRNAs encoding different isoforms of human Syk kinase can be found atGENBANK accession no. gi|21361552|ref|NM_(—)003177.2|,gi|496899|emb|Z29630.1|HSSYKPTK[496899] andgi|15030258|gb|BC011399.1|BC011399[15030258], which are incorporatedherein by reference.

Skilled artisans will appreciate that tyrosine kinases belonging toother families may have active sites or binding pockets that are similarin three-dimensional structure to that of Syk. As a consequence of thisstructural similarity, such kinases, referred to herein as “Syk mimics,”are expected to catalyze phosphorylation of substrates phosphorylated bySyk. Thus, it will be appreciated that such Syk mimics, signaltransduction cascades in which such Syk mimics play a role andbiological responses effected by such Syk mimics and Syk mimic-dependentsignaling cascades may be regulated, and in particular inhibited, withthe spiro 2,4-pyrimidinediamine compounds described herein.

“Syk-Dependent Signaling Cascade” refers to a signal transductioncascade in which Syk kinase plays a role. Non-limiting examples of suchSyk-dependent signaling cascades include the FcαRI, FcεRI, FcγRI,FcγRIII, BCR and integrin signaling cascades.

“Autoimmune Disease” refers to those diseases which are commonlyassociated with the nonanaphylactic hypersensitivity reactions (Type II,Type III and/or Type IV hypersensitivity reactions) that generallyresult as a consequence of the subject's own humoral and/orcell-mediated immune response to one or more immunogenic substances ofendogenous and/or exogenous origin. Such autoimmune diseases aredistinguished from diseases associated with the anaphylactic (Type I orIgE-mediated) hypersensitivity reaction.

6.3 Spiro 2,4-Pyrimidinediamine Compounds

Spiro 2,4-pyrimidinediamine compounds according to structural formula(I) are provided herein:

including salts, hydrates, solvates and N-oxides thereof, wherein:

-   -   L¹ is a direct bond or a linker;    -   L² is a direct bond or a linker;    -   R² is selected from the group consisting of (C1-C6) alkyl        optionally substituted with one or more of the same or different        R⁸ groups, (C3-C8) alkyl optionally substituted with one or more        of the same or different R⁸ groups, 3-8 membered        cycloheteroalkyl optionally substituted with one or more of the        same or different R⁸ groups, (C5-C15) aryl optionally        substituted with one or more of the same or different R⁸ groups,        phenyl optionally substituted with one or more of the same or        different R⁸ groups and 5-15 membered heteroaryl optionally        substituted with one or more of the same or different R⁸ groups;    -   R⁴ is

-   -   each W is, independently of the other, —CR³¹R³¹—;    -   X is selected from the group consisting of —N— and —CH—;    -   Y and Z are each, independently of one another, selected from        the group consisting of —O—, —S—, —SO—, —SO₂—, —SONR³⁶—, —NH—,        —NR³⁵— and —NR¹⁷—;    -   R⁵ is selected from the group consisting of hydrogen, an        electronegative group, —OR^(d), —SR^(d), (C1-C3) haloalkyloxy,        (C1-C3) perhaloalkyloxy, —NR^(c)R^(c), halogen, (C1-C3)        haloalkyl, (C1-C3) perhaloalkyl, —CF₃, —CH₂CF₃, —CF₂CF₃, —CN,        —NC, —OCN, —SCN, —NO, —NO₂, —N₃, —S(O)R^(d), —S(O)₂R^(d),        —S(O)₂OR^(d), —S(O)NR^(c)R^(c); —S(O)₂NR^(c)R^(c), —OS(O)R^(d),        —OS(O)₂R^(d), —OS(O)₂OR^(d), —OS(O)NR^(c)R^(c),        —OS(O)₂NR^(c)R^(c), —C(O)R^(d), —C(O)OR^(d), —C(O)NR^(c)R^(c),        —C(NH)NR^(c)R^(c), —OC(O)R^(d), —SC(O)R^(d), —OC(O)OR^(d),        —SC(O)OR^(d), —OC(O)NR^(c)R^(c), —SC(O)NR^(c)R^(c),        —OC(NH)NR^(c)R^(c), —SC(NH)NR^(c)R^(c), —[NHC(O)]_(n)R^(d),        —[NHC(O)]_(n)OR^(d), —[NHC(O)]_(n)NR^(c)R^(c) and        —[NHC(NH)]_(n)NR^(c)R^(c), (C5-C10) aryl optionally substituted        with one or more of the same or different R⁸ groups, phenyl        optionally substituted with one or more of the same or different        R⁸ groups, (C6-C16) arylalkyl optionally substituted with one or        more of the same or different R⁸ groups, 5-10 membered        heteroaryl optionally substituted with one or more of the same        or different R⁸ groups and 6-16 membered heteroarylalkyl        optionally substituted with one or more of the same or different        R⁸ groups, (C1-C6) alkyl optionally substituted with one or more        of the same or different R⁸ groups, (C1-C4) alkanyl optionally        substituted with one or more of the same or different R⁸ groups,        (C2-C4) alkenyl optionally substituted with one or more of the        same or different R⁸ groups and (C2-C4) alkynyl optionally        substituted with one or more of the same or different R⁸ groups;    -   R⁶ independently is selected from the group consisting of        hydrogen, an electronegative group, —OR^(d), —SR^(d), (C1-C3)        haloalkyloxy, (C1-C3) perhaloalkyloxy, —NR^(c)R^(c), halogen,        (C1-C3) haloalkyl, (C1-C3) perhaloalkyl, —CF₃, —CH₂CF₃, —CF₂CF₃,        —CN, —NC, —OCN, —SCN, —NO, —NO₂, —N₃, —S(O)R^(d), —S(O)₂R^(d),        —S(O)₂OR^(d), —S(O)NR^(c)R^(c); —S(O)₂NR^(c)R^(c), —OS(O)R^(d),        —OS(O)₂R^(d), —OS(O)₂OR^(d), —OS(O)NR^(c)R^(c),        —OS(O)₂NR^(c)R^(c), —C(O)R^(d), —C(O)OR^(d), —C(O)NR^(c)R^(c),        —C(NH)NR^(c)R^(c), —OC(O)R^(d), —SC(O)R^(d), —OC(O)OR^(d),        —SC(O)OR^(d), —OC(O)NR^(c)R^(c), —SC(O)NR^(c)R^(c),        —OC(NH)NR^(c)R^(c), —SC(NH)NR^(c)R^(c), —[NHC(O)]_(n)R^(d),        —[NHC(O)]_(n)OR^(d), —[NHC(O)]_(n)NR^(c)R^(c) and        —[NHC(NH)]_(n)NR^(c)R^(c), (C5-C10) aryl optionally substituted        with one or more of the same or different R⁸ groups, phenyl        optionally substituted with one or more of the same or different        R⁸ groups, (C6-C16) arylalkyl optionally substituted with one or        more of the same or different R⁸ groups, 5-10 membered        heteroaryl optionally substituted with one or more of the same        or different R⁸ groups and 6-16 membered heteroarylalkyl        optionally substituted with one or more of the same or different        R⁸ groups;

R⁸ is selected from the group consisting of R^(a), R^(b), R^(a)substituted with one or more of the same or different R^(a) or R^(b),—OR^(a) substituted with one or more of the same or different R^(a) orR^(b), —B(OR^(a))₂, —B(NR^(c)R^(c))₂, —(CH₂)_(m)—R^(b),—(CHR^(a))_(m)—R^(b), —O—(CH₂)_(m)—R^(b), —S—(CH₂)_(m)—R^(b),—O—CHR^(a)R^(b), —O—CR^(a)(R^(b))₂, —O—(CHR^(a))_(m)—R^(b), —O—(CH₂)_(m)—CH[(CH₂)_(m)R^(b)]R^(b), —S—(CHR^(a))_(m)—R^(b),—C(O)NH—(CH₂)_(m)—R^(b), — C(O)NH—(CHR^(a))_(m)—R^(b),—O—(CH₂)_(m)—C(O)NH—(CH₂)_(m)—R^(b),—S—(CH₂)_(m)—C(O)NH—(CH₂)_(m)—R^(b),—O—(CHR^(a))_(m)—C(O)NH—(CHR^(a))_(m)—R^(b),—S—(CHR^(a))_(m)—C(O)NH—(CHR^(a))_(m)—R^(b), —NH—(CH₂)_(m)—R^(b),—NH—(CHR^(a))_(m)—R^(b)— NH[(CH₂)_(m)R^(b)], —N[(CH₂)_(m)R^(b)]₂,—NHC(O)—NH—(CH₂)_(m)—R^(b), —NH—C(O)—(CH₂)_(m)—CHR^(b)R^(b) and—NH—(CH₂)_(m)—C(O)—NH—(CH₂)_(m)—R^(b);

-   -   each R³¹ is, independently of the others, hydrogen or (C1-C6)        alkyl optionally substituted with one or more of the same or        different R⁸ groups;    -   each R³⁵ is, independently of the other, selected from the group        consisting of hydrogen and R⁸, or, alternatively, the two R³⁵        groups are taken together to form an oxo (═O), or ═NR³⁸ group;    -   each R³⁶ is, independently of the others, selected from the        group consisting of hydrogen and (C1-C6) alkyl;    -   each R³⁷ is, independently of the others, selected from the        group consisting of hydrogen and a progroup;    -   R³⁸ is selected from the group consisting of hydrogen, (C1-C6)        alkyl and (C5-C14) aryl;    -   each R^(a) is, independently of the others, selected from the        group consisting of hydrogen, (C1-C6) alkyl, (C3-C8) cycloalkyl,        cyclohexyl, (C4-C11) cycloalkylalkyl, (C5-C10) aryl, phenyl,        (C6-C16) arylalkyl, benzyl, 2-6 membered heteroalkyl, 3-8        membered cycloheteroalkyl, morpholinyl, piperazinyl,        homopiperazinyl, piperidinyl, 4-11 membered        cycloheteroalkylalkyl, 5-10 membered heteroaryl and 6-16        membered heteroarylalkyl;    -   each R^(b) is, independently of the others, a suitable group        independently selected from the group consisting of ═O, —OR^(d),        (C1-C3) haloalkyloxy, —OCF₃, ═S, —SR, ═NR^(d), ═NOR^(d),        —NR^(c)R^(c), halogen, —CF₃, —CN, —NC, —OCN, —SCN, —NO, —NO₂,        ═N₂, —N₃, —S(O)R^(d), —S(O)₂R^(d), —S(O)₂OR^(d),        —S(O)NR^(c)R^(c), —S(O)₂NR^(c)R^(c), —OS(O)R^(d), —OS(O)₂R^(d),        —OS(O)₂OR^(d), —OS(O)₂NR^(c)R^(c), —C(O)R^(d), —C(O)OR^(d),        —C(O)NR^(c)R^(c), —C(NH)NR^(c)R^(c), —C(NR^(a))NR^(c)R^(c),        —C(NOH)R^(a), —C(NOH)NR^(c)R^(c), —OC(O)R^(d), —OC(O)OR^(d),        —OC(O)NR^(c)R^(c), —OC(NH)NR^(c)R^(c), —OC(NR^(a))NR^(c)R^(c),        —[NHC(O)]_(n)R^(d), —[NR^(a)C(O)]_(n)R^(d), —[NHC(O)]_(n)OR^(d),        —[NR^(a)C(O)]_(n)OR^(d) [NHC(O)]_(n)NR^(c)R^(c),        [NR^(a)C(O)]_(n)NR^(c)R^(c), —[NHC(NH)]_(n)NR^(c)R^(c) and        —[NR^(a)C(NR^(a))]_(n)NR^(c)R^(c);    -   each R^(c) is, independently or the others, a protecting group        or R^(a), or, alternatively, each R^(c) is taken together with        the nitrogen atom to which it is bonded to form a 5 to        8-membered cycloheteroalkyl or heteroaryl which may optionally        include one or more of the same or different additional        heteroatoms and which may optionally be substituted with one or        more of the same or different R^(a) or suitable R^(b) groups;    -   each R^(d) is, independently of the others, a protecting group        or R^(a);    -   each m is, independently of the others, an integer from 1 to 3;    -   each n is, independently of the others, an integer from 0 to 3;        and    -   o is an integer from 1 to 6.

In compounds of structural formula (I), L¹ and L² represent,independently of one another, a direct bond or a linker. Thus, as willbe appreciated by skilled artisans, the substituents R² and/or R⁴ may bebonded either directly to their respective nitrogen atoms or,alternatively, spaced away from their respective nitrogen atoms by wayof a linker. The identity of the linker is not critical and typicalsuitable linkers include, but are not limited to, (C1-C6) alkyldiyls,(C1-C6) alkenos and (C1-C6) heteroalkyldiyls, each of which may beoptionally substituted with one or more of the same or different R⁸groups, where R⁸ is as previously defined for structural formula (I). Insome embodiments, L¹ and L² are each, independently of one another,selected from the group consisting of a direct bond, (C1-C3) alkyldiyloptionally substituted with one or more of the same or different R^(a),suitable R^(b) or R⁹ groups and 1-3 membered heteroalkyldiyl optionallysubstituted with one or more of the same or different R^(a), suitableR^(b) or R⁹ groups, wherein R⁹ is selected from the group consisting of(C1-C3) alkyl, —OR^(a), —C(O)OR^(a), (C5-C10) aryl optionallysubstituted with one or more of the same or different halogens, phenyloptionally substituted with one or more of the same or differenthalogens, 5-10 membered heteroaryl optionally substituted with one ormore of the same or different halogens and 6 membered heteroaryloptionally substituted with one or more of the same or differenthalogens; and R^(a) and R^(b) are as previously defined for structuralformula (I). Specific R⁹ groups that may be used to substitute L¹ and L²include —OR^(a), —C(O)OR^(a), phenyl, halophenyl and 4-halophenyl,wherein R^(a) is as previously defined for structural formula (I).

In other embodiments, L¹ and L² are each, independently of one another,selected from the group consisting of methanol ethano and propano, eachof which may be optionally monosubstituted with an R⁹ group, where R⁹ isas previously defined above.

In the above embodiments, specific R^(a) groups that may be included inR⁹ groups are selected from the group consisting of hydrogen, (C₁-C₆)alkyl, phenyl and benzyl.

In still other embodiments, L¹ and L² are each a direct bond such thatthe spiro 2,4-pyrimidinediamine compounds are described by structuralformula (V):

including salts, hydrates, solvates and N-oxides thereof, wherein R²,R⁴, R⁵ and R⁶ are as previously defined for structural formula (I).Other embodiments of the spiro 2,4-pyrimidinediamine compounds ofstructural Formulae (I) and (V) are described below.

In some embodiments of compounds of structural Formulae (I) and (V), R⁵is halo, fluoro or —CF₃. In other embodiments, R⁵ is fluoro.

In some embodiments of compounds of structural Formulae (I) and (V), R⁶is hydrogen. In other embodiments, Y and Z are independently selectedfrom the group consisting of —O— and —NH—. In some other embodiments, Xis —CH—. In still other embodiments, Y is —O— and Z is —NH—.

In some embodiments of compounds of structural Formulae (I) and (V),each R³⁵ is hydrogen. In other embodiments, the two R³⁵ groups form anoxo group.

In some embodiments of compounds of structural Formulae (I) and (V), ois an integer from 1 to 4. In other embodiments, o is 1.

In some embodiments of structural Formulae (I) and (V), each R³¹ isindependently hydrogen or (C₁-C₆) alkyl. In other embodiments, each R³¹is hydrogen.

In some embodiments of structural Formulae (I) and (V), R² is phenyloptionally substituted with one or more of the same or different R⁵groups. In other embodiments, R² is a disubstituted phenyl group withtwo R^(b) groups or R² is a trisubstituted phenyl group with three R^(b)groups. In still other embodiments, R² is

In some embodiments of structural Formulae (I) and (V), R⁵ is halo,fluoro or —CF³ and R⁶ is hydrogen. In other embodiments, R⁵ is fluoroand R⁶ is hydrogen. In still other embodiments, the two R³⁵ groups forman oxo group, Y is O, Z is NH, X is CH and each R³¹ is hydrogen. Instill other embodiments, R⁵ is fluoro and R⁶ is hydrogen.

In some embodiments of structural Formulae (I) and (V), R² is phenyloptionally substituted with one or more of the same or different R⁸groups. In other embodiments, R² is a disubstituted phenyl group withtwo R^(b) groups or R² is a trisubstituted phenyl group with three R^(b)groups. In still other embodiments, R² is

In still other embodiments, o is 1. In still other embodiments, R2 isphenyl optionally substituted with one or more of the same or differentR8 groups. In still other embodiments, R2 is a disubstituted phenylgroup with two Rb groups or R2 is a trisubstituted phenyl group withthree R^(b) groups. In still other embodiments, R² is

Also specifically described are combinations of the above embodiments.

Those of skill in the art will appreciate that the spiro2,4-pyrimidinediamine compounds described herein may include functionalgroups that can be masked with progroups to create prodrugs. Suchprodrugs are usually, but need not be, pharmacologically inactive untilconverted into their active drug form. For example, ester groupscommonly undergo acid-catalyzed hydrolysis to yield the parentcarboxylic acid when exposed to the acidic conditions of the stomach, orbase-catalyzed hydrolysis when exposed to the basic conditions of theintestine or blood. Thus, when administered to a subject orally,2,4-pyrimidinediamines that include ester moieties may be consideredprodrugs of their corresponding carboxylic acid, regardless of whetherthe ester form is pharmacologically active.

In the prodrugs described herein, any available functional moiety may bemasked with a progroup to yield a prodrug. Functional groups within thespiro 2,4-pyrimidinediamine compounds that may be masked with progroupsfor inclusion in a promoiety include, but are not limited to, amines(primary and secondary), hydroxyls, sulfanyls (thiols), carboxyls, etc.Myriad progroups suitable for masking such functional groups to yieldpromoieties that are cleavable under the desired conditions of use areknown in the art. All of these progroups, alone or in combinations, maybe included in the prodrugs described herein.

In some, embodiments, the prodrugs are compounds according to structuralformula (I) in which R^(c) and R^(d) may be, in addition to theirpreviously-defined alternatives, a progroup.

Those of skill in the art will appreciate that many of the compounds andprodrugs described herein, as well as the various compound speciesspecifically described and/or illustrated herein, may exhibit thephenomena of tautomerism, conformational isomerism, geometric isomerismand/or optical isomerism. For example, the compounds and prodrugsdescribed herein may include one or more chiral centers and/or doublebonds and as a consequence may exist as stereoisomers, such asdouble-bond isomers (i.e., geometric isomers), enantiomers anddiastereomers and mixtures thereof, such as racemic mixtures. As anotherexample, the compounds and prodrugs described herein may exist inseveral tautomeric forms, including, for example, the enol form, theketo form and mixtures thereof. As the various compound names, formulaeand compound drawings within the specification and claims can representonly one of the possible tautomers, conformational isomers, opticalisomers or geometric isomers, it should be understood that the compoundsor prodrugs described herein include all possible tautomers,conformational isomers, optical isomers and/or geometric isomers, aswell as mixtures of these various different isomers. In cases of limitedrotation around the 2,4-pyrimidinediamine core structure, atropisomersare also possible and are also specifically included in the compoundsdescribed herein.

Moreover, skilled artisans will appreciate that when lists ofalternative substituents include members which, owing to valencyrequirements or other reasons, cannot be used to substitute a particulargroup, the list is intended to be read in context to include thosemembers of the list that are suitable for substituting the particulargroup. For example, skilled artisans will appreciate that while all ofthe listed alternatives for R^(b) can be used to substitute an alkylgroup, certain of the alternatives, such as ═O, cannot be used tosubstitute a phenyl group. It is to be understood that only possiblecombinations of substituent-group pairs are intended.

The compounds and/or prodrugs described herein may be identified byeither their chemical structure or their chemical name. When thechemical structure and the chemical name conflict, the chemicalstructure is determinative of the identity of the specific compound.

Depending upon the nature of the various substituents, the spiro2,4-pyrimidinediamine compounds and prodrugs described herein may be inthe form of salts. Such salts include salts suitable for pharmaceuticaluses (“pharmaceutically-acceptable salts”), salts suitable forveterinary uses, etc. Such salts may be derived from acids or bases, asis well-known in the art.

In some embodiments, the salt is a pharmaceutically acceptable salt.Generally, pharmaceutically acceptable salts are those salts that retainsubstantially one or more of the desired pharmacological activities ofthe parent compound and which are suitable for administration to humans.Pharmaceutically acceptable salts include acid addition salts formedwith inorganic acids or organic acids. Inorganic acids suitable forforming pharmaceutically acceptable acid addition salts include, by wayof example and not limitation, hydrohalide acids (e.g., hydrochloricacid, hydrobromic acid, hydriodic, etc.), sulfuric acid, nitric acid,phosphoric acid, and the like. Organic acids suitable for formingpharmaceutically acceptable acid addition salts include, by way ofexample and not limitation, acetic acid, trifluoroacetic acid, propionicacid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, oxalicacid, pyruvic acid, lactic acid, malonic acid, succinic acid, malicacid, maleic acid, fumaric acid, tartaric acid, citric acid, palmiticacid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid,mandelic acid, alkylsulfonic acids (e.g., methanesulfonic acid,ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonicacid, etc.), arylsulfonic acids (e.g., benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, cycloalkylsulfonic acids (e.g., camphorsulfonicacid), 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonicacid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylaceticacid, lauryl sulfuric acid, gluconic acid, glutamic acid,hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, andthe like.

Pharmaceutically acceptable salts also include salts formed when anacidic proton present in the parent compound is either replaced by ametal ion (e.g., an alkali metal ion, an alkaline earth metal ion or analuminum ion), an ammonium ion or coordinates with an organic base(e.g., ethanolamine, diethanolamine, triethanolamine, N-methylglucamine,morpholine, piperidine, dimethylamine, diethylamine, etc.).

The spiro 2,4-pyrimidinediamine compounds described herein as well asthe salts thereof, may also be in the form of hydrates, solvates andN-oxides, as are well-known in the art.

6.4 Methods of Synthesis

The compounds and prodrugs described may be synthesized via a variety ofdifferent synthetic routes using commercially available startingmaterials and/or starting materials prepared by conventional syntheticmethods. Suitable exemplary methods that may be routinely adapted tosynthesize 2,4-pyrimidinediamine compounds and prodrugs described hereinare found in U.S. Pat. No. 5,958,935, U.S. patent application Ser. No.10/355,543, filed Jan. 31, 2003 (U.S. Publication No. US20040029902-A1),International Publication No. WO 03/063794, U.S. patent application Ser.No. 10/631,029, filed Jul. 29, 2003 and International Publication No. WO2004/014382, published Feb. 19, 2004.

A variety of exemplary synthetic routes that can be used to synthesizespiro 2,4-pyrimidinediamine compounds are described in Schemes(I)-(VIII), below. In Schemes (I)-(VIII), like-numbered compounds havesimilar structures. These methods may be routinely adapted to synthesizethe prodrugs according to structural formula (II).

In one exemplary embodiment, the compounds can be synthesized fromsubstituted or unsubstituted uracils or thiouracils as illustrated inScheme (I), below:

In Scheme (I), R², R⁴, R⁵, R⁶, L¹ and L² are as previously defined forstructural formula (I), X is a halogen (e.g., F, Cl, Br or J) and Y andY′ are each, independently of one another, selected from the groupconsisting of O and S. Referring to Scheme (I), uracil or thiouracil 2is dihalogenated at the 2- and 4-positions using standard halogenatingagent POX₃ (or other standard halogenating agent) under standardconditions to yield 2,4-bishalo pyrimidine 4. Depending upon the R⁵substituent, in pyrimidine 4, the halide at the C4 position is morereactive towards nucleophiles than the halide at the C2 position. Thisdifferential reactivity can be exploited to synthesize2,4-pyrimidinediamines according to structural formula (I) by firstreacting 2,4-bishalopyrimidine 4 with one equivalent of amine 10,yielding 4N-substituted-2-halo-4-pyrimidineamine 8, followed by amine 6to yield a 2,4-pyrimidinediamine according structural formula (I).

In most situations, the C4 halide is more reactive towards nucleophiles,as illustrated in the Scheme. However, as will be recognized by skilledartisans, the identity of the R⁵ substituent may alter this reactivity.For example, when R⁵ is trifluoromethyl, a 50:50 mixture of4N-substituted-4-pyrimidineamine 8 and the corresponding2N-substituted-2-pyrimidineamine is obtained. Regardless of the identityof the R⁵ substituent, the regioselectivity of the reaction can becontrolled by adjusting the solvent and other synthetic conditions (suchas temperature), as is well-known in the art.

The reactions depicted in Scheme (I) may proceed more quickly when thereaction mixtures are heated via microwave. When heating in thisfashion, the following conditions may be used: heat to 175° C. inethanol for 5-20 min. in a Smith Reactor (Personal Chemistry) in asealed tube (at 20 bar pressure).

The uracil or thiouracil 2 starting materials may be purchased fromcommercial sources or prepared using standard techniques of organicchemistry. Commercially available uracils and thiouracils that can beused as starting materials in Scheme (I) include, by way of example andnot limitation, uracil (Aldrich #13,078-8; CAS Registry 66-22-8);2-thio-uracil (Aldrich #11,558-4; CAS Registry 141-90-2);2,4-dithiouracil (Aldrich #15, 846-1; CAS Registry 2001-93-6);5-acetouracil (Chem. Sources Int'l 2000; CAS Registry 6214-65-9);5-azidouracil; 5-aminouracil (Aldrich #85, 528-6; CAS Registry932-52-5); 5-bromouracil (Aldrich #85, 247-3; CAS Registry 51-20-7);5-(trans-2-bromovinyl)-uracil (Aldrich #45, 744-2; CAS Registry69304-49-0); 5-(trans-2-chlorovinyl)-uracil (CAS Registry 81751-48-2);5-(trans-2-carboxyvinyl)-uracil; uracil-5-carboxylic acid(2,4-dihydroxypyrimidine-5-carboxylic acid hydrate; Aldrich #27, 770-3;CAS Registry 23945-44-0); 5-chlorouracil (Aldrich #22, 458-8; CASRegistry 1820-81-1); 5-cyanouracil (Chem. Sources Int'l 2000; CASRegistry 4425-56-3); 5-ethyluracil (Aldrich #23,044-8; CAS Registry4212-49-1); 5-ethenyluracil (CAS Registry 37107-81-6); 5-fluorouracil(Aldrich #85, 847-1; CAS Registry 51-21-8); 5-iodouracil (Aldrich #85,785-8; CAS Registry 696-07-1); 5-methyluracil (thymine; Aldrich #13,199-7; CAS Registry 65-71-4); 5-nitrouracil (Aldrich #85, 276-7; CASRegistry 611-08-5); uracil-5-sulfamic acid (Chem. Sources Int'l 2000;CAS Registry 5435-16-5); 5-(trifluoromethyl)-uracil (Aldrich #22, 327-1;CAS Registry 54-20-6); 5-(2,2,2-trifluoroethyl)-uracil (CAS Registry155143-31-6); 5-(pentafluoroethyl)-uracil (CAS Registry 60007-38-3);6-aminouracil (Aldrich #A5060-6; CAS Registry 873-83-6)uracil-6-carboxylic acid (orotic acid; Aldrich #0-840-2; CAS Registry50887-69-9); 6-methyluracil (Aldrich #D11,520-7; CAS Registry 626-48-2);uracil-5-amino-6-carboxylic acid (5-aminoorotic acid; Aldrich #19,121-3; CAS Registry #7164-43-4); 6-amino-5-nitrosouracil(6-amino-2,4-dihydroxy-5-nitrosopyrimidine; Aldrich #27, 689-8; CASRegistry 5442-24-0); uracil-5-fluoro-6-carboxylic acid (5-fluorooroticacid; Aldrich #42, 513-3; CAS Registry 00000-00-0); anduracil-5-nitro-6-carboxylic acid (5-nitroorotic acid; Aldrich #18,528-0; CAS Registry 600779-49-9). Additional 5-, 6- and 5,6-substituteduracils and/or thiouracils are available from General Intermediates ofCanada, Inc., Edmonton, Alberta, CA and/or Interchim, France or may beprepared using standard techniques. Myriad textbook references teachingsuitable synthetic methods are provided infra.

Amines 10 may be purchased from commercial sources or, alternatively,may be synthesized utilizing standard techniques. For example, suitableamines may be synthesized from nitro precursors using standardchemistry. See also Vogel, 1989, Practical Organic Chemistry, AddisonWesley Longman, Ltd. and John Wiley & Sons, Inc. Amines 6 may besynthesized as described, infra, in Schemes (VII) and (VIII).

A specific embodiment of Scheme (I) utilizing 5-fluorouracil (Aldrich#32, 937-1) as a starting material is illustrated in Scheme (Ia), below:

In Scheme (Ia), R², R⁴, L¹ and L² are as previously defined for Scheme(I). According to Scheme (Ia), 5-fluorouracil 3 is halogenated withPOCl₃ to yield 2,4-dichloro-5-fluoropyrimidine 5. Reaction of2,4-dichloro-5-fluoropyrimidine 5 with one equivalent of amine 10 (toyield 2-chloro-N4-substituted-5-fluoro-4-pyrimidineamine 7) followed byone or more equivalents of amine 6.

In still another exemplary embodiment, the 2,4-pyrimidinediaminecompounds of the invention may be synthesized from substituted orunsubstituted 2-amino-4-pyrimidinols as illustrated in Scheme (II),below:

In Scheme (II), R², R⁴, R⁵, R⁶, L¹, L² and X are as previously definedfor Scheme (I) and Z is a leaving group as discussed in more detail inconnection with Scheme III, infra. Referring to Scheme (II),2-amino-4-pyrimidinol 30 is reacted with amine 6 (or optionallyprotected amine 21) to yield N2-substituted-4-pyrimidinol 32, which isthen halogenated as previously described to yieldN2-substituted-4-halo-2-pyrimidineamine 34. Optional deprotection (forexample if protected amine 21 was used in the first step) followed byreaction with amine 10 affords a 2,4-pyrimidinediamine according tostructural formula (I). Alternatively, pyrimidinol 30 can be reactedwith acylating agent 31.

Suitable commercially-available 2-amino-4-pyrimidinols 30 that can beused as starting materials in Scheme (III) include, but are not limitedto, 2-amino-6-chloro-4-pyrimidinol hydrate (Aldrich #A4702-8; CASRegistry 00000-00-0) and 2-amino-6-hydroxy-4-pyrimidinol (Aldrich#A5040-1; CAS Registry 56-09-7). Other 2-amino-4-pyrimidinols 30 usefulas starting materials in Scheme (III) are available from GeneralIntermediates of Canada, Inc., Edmonton, Alberta, CA and/or Interchim,France or may be prepared using standard techniques. Myriad textbookreferences teaching suitable synthetic methods are provided infra.

In still another exemplary embodiment, the 2,4-pyrimidinediaminecompounds of the invention can be prepared from2-chloro-4-aminopyrimidines or 2-amino-4-chloropyrimidines asillustrated in Scheme (III), below:

In Scheme (III), R², R⁴, R⁵, R⁶, L¹ and L² are as defined for Scheme (I)and Z is a leaving groups such as, for example, a halogen,methanesulfonyloxy, trifluoromethanesulfonyloxy, p-toluenesulfonyloxy,benzenesulfonyloxy, etc. Referring to Scheme (III),2-amino-4-chloropyrimidine 50 is reacted with amino 10 to yield4N-substituted-2-pyrimidineamine 52 which, following reaction withcompound 31 or amine 6, yields a 2,4-pyrimidinediamine according tostructural formula (I). Alternatively, 2-chloro-4-amino-pyrimidine 54may be reacted with compound 44 followed by amine 6 to yield a compoundaccording to structural formula (I).

A variety of pyrimidines 50 and 54 suitable for use as startingmaterials in Scheme (V) are commercially available, including by way ofexample and not limitation, 2-amino-4,6-dichloropyrimidine (Aldrich#A4860-1; CAS Registry 56-05-3); 2-amino-4-chloro-6-methoxy-pyrimidine(Aldrich #51, 864-6; CAS Registry 5734-64-5);2-amino-4-chloro-6-methylpyrimidine (Aldrich #12, 288-2; CAS Registry5600-21-5); and 2-amino-4-chloro-6-methylthiopyrimidine (Aldrich#A4600-5; CAS Registry 1005-38-5). Additional pyrimidine startingmaterials are available from General Intermediates of Canada, Inc.,Edmonton, Alberta, CA and/or Interchim, France or may be prepared usingstandard techniques. Myriad textbook references teaching suitablesynthetic methods are provided infra.

Alternatively, 4-chloro-2-pyrimidineamines 50 may be prepared asillustrated in Scheme (IV):

In Scheme (IV), R⁵ and R⁶ are as previously defined for structuralformula (I). In Scheme (IV), dicarbonyl 53 is reacted with guanidine toyield 2-pyrimidineamine 51. Reaction with peracids likem-chloroperbenzoic acid, trifluoroperacetic acid or urea hydrogenperoxide complex yields N-oxide 55, which is then halogenated to give4-chloro-2-pyrimidineamine 50. The corresponding4-halo-2-pyrimidineamines may be obtained by using suitable halogenationreagents.

As will be recognized by skilled artisans, spiro 2,4-pyrimidinediamines,synthesized via the exemplary methods described above or by otherwell-known means, may also be utilized as starting materials and/orintermediates to synthesize additional spiro 2,4-pyrimidinediaminecompounds. A specific example is illustrated in Scheme (V), below:

In Scheme (V), R4, R5, R6, L2 and Ra are as previously defined forstructural formula (I). Each Ra′ is independently an Ra, and may be thesame or different from the illustrated Ra. Referring to Scheme (V),carboxylic acid or ester 100 may be converted to amide 104 by reactionwith amine 102. In amine 102, Ra may be the same or different than Ra ofacid or ester 100. Similarly, carbonate ester 106 may be converted tocarbamate 108.

A second specific example is illustrated in Scheme (VI), below:

In Scheme (VI), R⁴, R⁵, R⁶, L² and R^(c) are as previously defined forstructural formula (I). Referring to Scheme (VI), amide 110 or 116 maybe converted to amine 114 or 118, respectively, by borane reduction withborane methylsulfide complex 112. Other suitable reactions forsynthesizing spiro 2,4-pyrimidinediamine compounds from spiro2,4-pyrimidinediamine starting materials will be apparent to those ofskill in the art.

Scheme (VII) describes the preparation of spiro heterocylic aryl aminescorresponding to compound 10 (i.e., R⁴-L²-NH₂). In Scheme (VII), infra,U is —SH, —NHR^(e) or —OH, V is —NO₂, —NHR^(e), —OR^(e) or —SR^(e), D ishydrogen, —NO₂, or NHR^(e), each R^(e) is independently a protectinggroup or hydrogen, B is a leaving group such as halogen, mesylate,tosylate, etc., T and P are independently —NR^(e)—, —S—, or —O—, each Mis independently a leaving group such as halogen, mesylate, tosylate,etc. or OR^(e), R³¹, R³⁵, X, Y, Z and o are as previously defined,supra.

Referring to Scheme (VII), compound 120, is alkylated with ester 132 toprovide after intramolecular cyclization, the annelation product 134.Alkylation of 134 with bifunctional agent 124 provides the monoalkylproduct 136 which may be intramolecularly cyclized to yield 130 whichmay be converted to compound 132 by conventional methods known to theskilled artisan. For example, sulfides (i.e., where T and/or P aresulfur) and amines (i.e., where T and/or P are nitrogen) may beconverted to sulfoxides, sulforates, sulfonamides, alkylamines,arylamines and protected amines by routine synthetic methods. Thevarious embodiments of substituent D of compound 130 may beinterconverted, as H may be converted to NO₂ by nitration, NO₂ to NH₂ byreduction, etc. Finally, as is also known in the art, the carbonyl groupin compound 130 may be converted to the dihydro compound by reductionwith conventional reagents (e.g., lithium aluminum hydride). In someembodiments of compound 124, one M is halogen and the other M is OR^(e).Accordingly in some embodiments, group M in compound 136 is either aleaving group or may be converted to a leaving group (i.e., when M isOR^(e)) by conventional methods.

Although many of the synthetic schemes discussed above do not illustratethe use of protecting groups, skilled artisans will recognize that insome instances substituents R², R⁴, R⁵, R⁶, L¹ and/or L² may includefunctional groups requiring protection. The exact identity of theprotecting group used will depend upon, among other things, the identityof the functional group being protected and the reaction conditions usedin the particular synthetic scheme, and will be apparent to those ofskill in the art. Guidance for selecting protecting groups andchemistries for their attachment and removal suitable for a particularapplication can be found, for example, in Greene & Wuts, ProtectiveGroups in Organic Synthesis, 3d Edition, John Wiley & Sons, Inc., NewYork (1999).

Prodrugs according to structural formula (II) may be prepared by routinemodification of the above-described methods. Alternatively, suchprodrugs may be prepared by reacting a suitably protected2,4-pyrimidinediamine of structural formula (I) with a suitableprogroup. Conditions for carrying out such reactions and fordeprotecting the product to yield a prodrug of formula (II) arewell-known.

Myriad references teaching methods useful for synthesizing pyrimidinesgenerally, as well as starting materials described in Schemes(I)-(VIII), are known in the art. For specific guidance, the reader isreferred to Brown, D. J., “The Pyrimidines”, in The Chemistry ofHeterocyclic Compounds, Volume 16 (Weissberger, A., Ed.), 1962,Interscience Publishers, (A Division of John Wiley & Sons), New York(“Brown I”); Brown, D. J., “The Pyrimidines”, in The Chemistry ofHeterocyclic Compounds, Volume 16, Supplement I (Weissberger, A. andTaylor, E. C., Ed.), 1970, Wiley-Interscience, (A Division of John Wiley& Sons), New York (Brown II”); Brown, D. J., “The Pyrimidines”, in TheChemistry of Heterocyclic Compounds, Volume 16, Supplement II(Weissberger, A. and Taylor, E. C., Ed.), 1985, An IntersciencePublication (John Wiley & Sons), New York (“Brown III”); Brown, D. J.,“The Pyrimidines” in The Chemistry of Heterocyclic Compounds, Volume 52(Weissberger, A. and Taylor, E. C., Ed.), 1994, John Wiley & Sons, Inc.,New York, pp. 1-1509 (Brown IV”); Kenner, G. W. and Todd, A., inHeterocyclic Compounds, Volume 6, (Elderfield, R. C., Ed.), 1957, JohnWiley, New York, Chapter 7 (pyrimidines); Paquette, L. A., Principles ofModern Heterocyclic Chemistry, 1968, W. A. Benjamin, Inc., New York, pp.1-401 (uracil synthesis pp. 313, 315; pyrimidine synthesis pp. 313-316;amino pyrimidine synthesis pp. 315); Joule, J. A., Mills, K. and Smith,G. F., Heterocyclic Chemistry, 3^(rd) Edition, 1995, Chapman and Hall,London, UK, pp. 1-516; Vorbruiggen, H. and Ruh-Pohlenz, C., Handbook ofNucleoside Synthesis, John Wiley & Sons, New York, 2001, pp. 1-631(protection of pyrimidines by acylation pp. 90-91; silylation ofpyrimidines pp. 91-93); Joule, J. A., Mills, K. and Smith, G. F.,Heterocyclic Chemistry, 4^(th) Edition, 2000, Blackwell Science, Ltd,Oxford, UK, pp. 1-589; and Comprehensive Organic Synthesis, Volumes 1-9(Trost, B. M. and Fleming, I., Ed.), 1991, Pergamon Press, Oxford, UK.

It should be understood by the skilled artisan that in Schemes I throughVIII, the N4 nitrogen can be substituted by R^(4c) as describedthroughout the specification and in the examples provided herein.

6.5 Inhibition of Fc Receptor Signal Cascades

Active spiro 2,4-pyrimidinediamine compounds described herein mayinhibit Fc receptor signaling cascades that lead to, among other things,degranulation of cells. As a specific example, the compounds inhibit theFcεRI and/or FcγRI signal cascades that lead to degranulation of immunecells such as neutrophil, eosinophil, mast and/or basophil cells. Bothmast and basophil cells play a central role in allergen-induceddisorders, including, for example, allergic rhinitis and asthma.Referring to FIG. 1, upon exposure allergens, which may be, among otherthings, pollen or parasites, allergen-specific IgE antibodies aresynthesized by B-cells activated by IL-4 (or IL-13) and other messengersto switch to IgE class specific antibody synthesis. Theseallergen-specific IgEs bind to the high affinity FcεRI. Upon binding ofantigen, the FcεR1-bound IgEs are cross-linked and the IgE receptorsignal transduction pathway is activated, which leads to degranulationof the cells and consequent release and/or synthesis of a host ofchemical mediators, including histamine, proteases (e.g., tryptase andchymase), lipid mediators such as leukotrienes (e.g., LTC4),platelet-activating factor (PAF) and prostaglandins (e.g., PGD2) and aseries of cytokines, including TNF-α, IL-4, IL-13, IL-5, IL-6, IL-8,GMCSF, VEGF and TGF-β. The release and/or synthesis of these mediatorsfrom mast and/or basophil cells accounts for the early and late stageresponses induced by allergens, and is directly linked to downstreamevents that lead to a sustained inflammatory state.

The molecular events in the FcεRI signal transduction pathway that leadto release of preformed mediators via degranulation and release and/orsynthesis of other chemical mediators are well-known and are illustratedin FIG. 2. Referring to FIG. 2, the FcεRI is a heterotetrameric receptorcomposed of an IgE-binding alpha-subunit, a beta subunit, and two gammasubunits (gamma homodimer). Cross-linking of FcεRI-bound IgE bymultivalent binding agents (including, for example IgE-specificallergens or anti-IgE antibodies or fragments) induces the rapidassociation and activation of the Src-related kinase Lyn. Lynphosphorylates immunoreceptor tyrosine-based activation motifs (ITAMS)on the intracellular beta and gamma subunits, which leads to therecruitment of additional Lyn to the beta subunit and Syk kinase to thegamma homodimer. These receptor-associated kinases, which are activatedby intra- and intermolecular phosphorylation, phosphorylate othercomponents of the pathway, such as the Btk kinase, LAT, andphospholipase C-gamma PLC-gamma). Activated PLC-gamma initiates pathwaysthat lead to protein kinase C activation and Ca²⁺ mobilization, both ofwhich are required for degranulation. FcεR1 cross-linking also activatesthe three major classes of mitogen activated protein (MAP) kinases, i.e.ERK1/2, JNK1/2, and p38. Activation of these pathways is important inthe transcriptional regulation of proinflammatory mediators, such asTNF-α and IL-6, as well as the lipid mediator leukotriene CA (LTC4).

Although not illustrated, the FcγRI signaling cascade is believed toshare some common elements with the FceRI signaling cascade.Importantly, like FcεRI, the FcγRI includes a gamma homodimer that isphosphorylated and recruits Syk, and like FcεRI, activation of the FcγRIsignaling cascade leads to, among other things, degranulation. Other Fcreceptors that share the gamma homodimer, and which can be regulated bythe active 2,4-pyrimidinediamine compounds include, but are not limitedto, FcαRI and FcγRIII.

The ability of spiro 2,4-pyrimidinediamine compounds to inhibit Fcreceptor signaling cascades may be simply determined or confirmed in invitro assays. Suitable assays for confirming inhibition ofFcεRI-mediated degranulation are provided in the Examples section. Inone typical assay, cells capable of undergoing FcεRI-mediateddegranulation, such as mast or basophil cells, are first grown in thepresence of IL-4, Stem Cell Factor (SCF), IL-6 and IgE to increaseexpression of the FcεRI, exposed to a 2,4-pyrimidinediamine testcompound of the invention and stimulated with anti-IgE antibodies (or,alternatively, an IgE-specific allergen). Following incubation, theamount of a chemical mediator or other chemical agent released and/orsynthesized as a consequence of activating the FcεRI signaling cascademay be quantified using standard techniques and compared to the amountof the mediator or agent released from control cells (i.e., cells thatare stimulated but that are not exposed to test compound). Theconcentration of test compound that yields a 50% reduction in thequantity of the mediator or agent measured as compared to control cellsis the IC₅₀ of the test compound. The origin of the mast or basophilcells used in the assay will depend, in part, on the desired use for thecompounds and will be apparent to those of skill in the art. Forexample, if the compounds will be used to treat or prevent a particulardisease in humans, a convenient source of mast or basophil cells is ahuman or other animal which constitutes an accepted or known clinicalmodel for the particular disease. Thus, depending upon the particularapplication, the mast or basophil cells may be derived from a widevariety of animal sources, ranging from, for example, lower mammals suchas mice and rats, to dogs, sheep and other mammals commonly employed inclinical testing, to higher mammals such as monkeys, chimpanzees andapes, to humans. Specific examples of cells suitable for carrying outthe in vitro assays include, but are not limited to, rodent or humanbasophil cells, rat basophil leukemia cell lines, primary mouse mastcells (such as bone marrow-derived mouse mast cells “BMMC”) and primaryhuman mast cells isolated from cord blood (“CHMC”) or other tissues suchas lung. Methods for isolating and culturing these cell types arewell-known or are provided in the Examples section (see, e.g., Demo etal., 1999, Cytometry 36(4):340-348 and copending U.S. application Ser.No. 10/053,355, filed Nov. 8, 2001, the disclosures of which areincorporated herein by reference). Of course, other types of immunecells that degranulate upon activation of the FcεRI signaling cascademay also be used, including, for example, eosinophils.

As will be recognized by skilled artisans, the mediator or agentquantified is not critical. The only requirement is that it be amediator or agent released and/or synthesized as a consequence ofinitiating or activating the Fc receptor signaling cascade. For example,referring to FIG. 1, activation of the FcεRI signaling cascade in mastand/or basophil cells leads to numerous downstream events. For example,activation of the FcεRI signal cascade leads to the immediate release(i.e., within 1-3 min. following receptor activation) of a variety ofpreformed chemical mediators and agents via degranulation. Thus, in oneembodiment, the mediator or agent quantified may be specific to granules(i.e., present in granules but not in the cell cytoplasm generally).Examples of granule-specific mediators or agents that can be quantifiedto determine and/or confirm the activity of a 2,4-pyrimidinediaminecompound of the invention include, but are not limited to,granule-specific enzymes such as hexosaminidase and tryptase andgranule-specific components such as histamine and serotonin. Assays forquantifying such factors are well-known, and in many instances arecommercially available. For example, tryptase and/or hexosaminidaserelease may be quantified by incubating the cells with cleavablesubstrates that fluoresce upon cleavage and quantifying the amount offluorescence produced using conventional techniques. Such cleavablefluorogenic substrates are commercially available. For example, thefluorogenic substrates Z-Gly-Pro-Arg-AMC (Z=benzyloxycarbonyl;AMC=7-amino-4-methylcoumarin; BIOMOL Research Laboratories, Inc.,Plymouth Meeting, Pa. 19462, Catalog No. P-142) and Z-Ala-Lys-Arg-AMC(Enzyme Systems Products, a division of ICN Biomedicals, Inc.,Livermore, Calif. 94550, Catalog No. AMC-246) can be used to quantifythe amount of tryptase released. The fluorogenic substrate4-methylumbelliferyl-N-acetyl-β-D-glucosaminide (Sigma, St. Louis, Mo.,Catalog #69585) can be used to quantify the amount of hexosaminidasereleased. Histamine release may be quantified using a commerciallyavailable enzyme-linked immunosorbent assay (ELISA) such as Immunotechhistamine ELISA assay #IM2015 (Beckman-Coulter, Inc.). Specific methodsof quantifying the release of tryptase, hexosaminidase and histamine areprovided in the Examples section. Any of these assays may be used todetermine or confirm the activity of spiro 2,4-pyrimidinediamine.

Referring again to FIG. 1, degranulation is only one of severalresponses initiated by the FcεRI signaling cascade. In addition,activation of this signaling pathway leads to the de novo synthesis andrelease of cytokines and chemokines such as IL-4, IL-5, IL-6, TNF-α,IL-13 and MIP1-α), and release of lipid mediators such as leukotrienes(e.g., LTC4), platelet activating factor (PAF) and prostaglandins.Accordingly, the 2,4-pyrimidinediamine compounds of the invention mayalso be assessed for activity by quantifying the amount of one or moreof these mediators released and/or synthesized by activated cells.

Unlike the granule-specific components discussed above, these “latestage” mediators are not released immediately following activation ofthe FcεRI signaling cascade. Accordingly, when quantifying these latestage mediators, care should be taken to insure that the activated cellculture is incubated for a time sufficient to result in the synthesis(if necessary) and release of the mediator being quantified. Generally,PAF and lipid mediators such as leukotriene C4 are released 3-30 min.following FcεRI activation. The cytokines and other late stage mediatorsare released approx. 4-8 hrs. following FcεRI activation. Incubationtimes suitable for a specific mediator will be apparent to those ofskill in the art. Specific guidance and assays are provided in theExamples section.

The amount of a particular late stage mediator released may bequantified using any standard technique. In one embodiment, theamount(s) may be quantified using ELISA assays. ELISA assay kitssuitable for quantifying the amount of TNFα, IL-4, IL-5, IL-6 and/orIL-13 released are available from, for example, Biosource International,Inc., Camarillo, Calif. 93012 (see, e.g., Catalog Nos. KHC3011, KHC0042,KHC0052, KHC0061 and KHC0132). ELISA assay kits suitable for quantifyingthe amount of leukotriene C4 (LTC4) released from cells are availablefrom Cayman Chemical Co., Ann Arbor, Mich. 48108 (see, e.g., Catalog No.520211).

Typically, active spiro 2,4-pyrimidinediamine compounds will exhibitIC₅₀s with respect to FcεRI-mediated degranulation and/or mediatorrelease or synthesis of about 20 μM or lower, as measured in an in vitroassay, such as one of the in vitro assays described above or in theExamples section. Of course, skilled artisans will appreciate thatcompounds which exhibit lower IC₅₀s, for example on the order of 10 μM,1 μM, 100 nM, 10 nM, 1 nM, or even lower, are particularly useful.

Skilled artisans will also appreciate that the various mediatorsdiscussed above may induce different adverse effects or exhibitdifferent potencies with respect to the same adverse effect. Forexample, the lipid mediator LTC4 is a potent vasoconstrictor—it isapproximately 1000-fold more potent at inducing vasoconstriction thanhistamine. As another example, in addition to mediating atopic or Type Ihypersensitivity reactions, cytokines can also cause tissue remodelingand cell proliferation. Thus, although compounds that inhibit releaseand/or synthesis of any one of the previously discussed chemicalmediators are useful, skilled artisans will appreciate that compoundswhich inhibit the release and/or synthesis of a plurality, or even all,of the previously described mediators find particular use, as suchcompounds are useful for ameliorating or avoiding altogether aplurality, or even all, of the adverse effects induced by the particularmediators. For example, compounds which inhibit the release of all threetypes of mediators-granule-specific, lipid and cytokine are useful fortreating or preventing immediate Type I hypersensitivity reactions aswell as the chronic symptoms associated therewith.

Compounds described herein capable of inhibiting the release of morethan one type of mediator (e.g., granule-specific or late stage) may beidentified by determining the IC₅₀ with respect to a mediatorrepresentative of each class using the various in vitro assays describedabove (or other equivalent in vitro assays). Compounds which are capableof inhibiting the release of more than one mediator type will typicallyexhibit an IC₅₀ for each mediator type tested of less than about 20 μM.For example, a compound which exhibits an IC₅₀ of 1 μM with respect tohistamine release (IC₅₀ ^(histamine)) and an IC₅₀ of 1 nM with respectto leukotriene LTC4 synthesis and/or release (IC₅₀ ^(LTC4)) inhibitsboth immediate (granule-specific) and late stage mediator release. Asanother specific example, a compound that exhibits an IC₅₀ ^(tryptase)of 10 μM, an IC₅₀ ^(LTC4) of 1 μM and an IC₅₀ ^(IL-4) of 1 μM inhibitsimmediate (granule-specific), lipid and cytokine mediator release.Although the above specific examples utilize the IC₅₀s of onerepresentative mediator of each class, skilled artisans will appreciatethat the IC₅₀s of a plurality, or even all, mediators comprising one ormore of the classes may be obtained. The quantity(ies) and identity(ies)of mediators for which IC₅₀ data should be ascertained for a particularcompound and application will be apparent to those of skill in the art.

Similar assays may be utilized to confirm inhibition of signaltransduction cascades initiated by other Fc receptors, such as FcαRI,FcγRI and/or FcγRIII signaling, with routine modification. For example,the ability of the compounds to inhibit FcγRI signal transduction may beconfirmed in assays similar to those described above, with the exceptionthat the FcγRI signaling cascade is activated, for example by incubatingthe cells with IgG and an IgG-specific allergen or antibody, instead ofIgE and an IgE-specific allergen or antibody. Suitable cell types,activating agents and agents to quantify to confirm inhibition of otherFc receptors, such as Fc receptors that comprise a gamma homodimer, willbe apparent to those of skill in the art.

One particularly useful class of compounds includes those spiro2,4-pyrimidinediamine compounds that inhibit the release of immediategranule-specific mediators and late stage mediators with approximatelyequivalent IC₅₀s. By approximately equivalent is meant that the IC₅₀sfor each mediator type are within about a 10-fold range of one another.Another particularly useful class of compounds includes those spiro2,4-pyrimidinediamine compounds that inhibit the release of immediategranule-specific mediators, lipid mediators and cytokine mediators withapproximately equivalent IC₅₀s. In a specific embodiment, such compoundsinhibit the release of the following mediators with approximatelyequivalent IC₅₀s: histamine, tryptase, hexosaminidase, IL-4, IL-5, IL-6,IL-13, TNFα and LTC4. Such compounds are particularly useful for, amongother things, ameliorating or avoiding altogether both the early andlate stage responses associated with atopic or immediate Type Ihypersensitivity reactions.

Ideally, the ability to inhibit the release of all desired types ofmediators will reside in a single compound. However, mixtures ofcompounds can also be identified that achieve the same result. Forexample, a first compound which inhibits the release of granule specificmediators may be used in combination with a second compound whichinhibits the release and/or synthesis of cytokine mediators.

In addition to the FcεRI or FcγRI degranulation pathways discussedabove, degranulation of mast and/or basophil cells can be induced byother agents. For example, ionomycin, a calcium ionophore that bypassesthe early FcεRI or FcγRI signal transduction machinery of the cell,directly induces a calcium flux that triggers degranulation. Referringagain to FIG. 2, activated PLCγ initiates pathways that lead to, amongother things, calcium ion mobilization and subsequent degranulation. Asillustrated, this Ca²⁺ mobilization is triggered late in the FcεRIsignal transduction pathway. As mentioned above, and as illustrated inFIG. 3, ionomycin directly induces Ca²⁺ mobilization and a Ca²⁺ fluxthat leads to degranulation. Other ionophores that induce degranulationin this manner include A23187. The ability of granulation-inducingionophores such as ionomycin to bypass the early stages of the FcεRIand/or FcγRI signaling cascades may be used as a counter screen toidentify active compounds of the invention that specifically exert theirdegranulation-inhibitory activity by blocking or inhibiting the earlyFcεRI or FcγRI signaling cascades, as discussed above. Compounds whichspecifically inhibit such early FcεRI or FcγRI-mediated degranulationinhibit not only degranulation and subsequent rapid release ofhistamine, tryptase and other granule contents, but also inhibit thepro-inflammatory activation pathways causing the release of TNFα, IL-4,IL-13 and the lipid mediators such as LTC4. Thus, compounds whichspecifically inhibit such early FcεRI and/or FcγRI-mediateddegranulation block or inhibit not only acute atopic or Type Ihypersensitivity reactions, but also late responses involving multipleinflammatory mediators.

Compounds described herein that specifically inhibit early FcεRI and/orFcγRI-mediated degranulation are those compounds that inhibit FcεRIand/or FcγRI-mediated degranulation (for example, have an IC₅₀ of lessthan about 20 μM with respect to the release of a granule-specificmediator or component as measured in an in vitro assay with cellsstimulated with an IgE or IgG binding agent) but that do not appreciablyinhibit ionophore-induced degranulation. In one embodiment, compoundsare considered to not appreciably inhibit ionophore-induceddegranulation if they exhibit an IC₅₀ of ionophore-induced degranulationof greater than about 20 μM, as measured in an in vitro assay. Ofcourse, active compounds that exhibit even higher IC₅₀'s ofionophore-induced degranulation, or that do not inhibitionophore-induced degranulation at all, are particularly useful. Inanother embodiment, compounds are considered to not appreciably inhibitionophore-induced degranulation if they exhibit a greater than 10-folddifference in their IC₅₀s of FcεRI and/or FcγRI-mediated degranulationand ionophore-induced degranulation, as measured in an in vitro assay.Assays suitable for determining the IC₅₀ of ionophore-induceddegranulation include any of the previously-described degranulationassays, with the modification that the cells are stimulated or activatedwith a degranulation-inducing calcium ionophore such as ionomycin orA23187 (A.G. Scientific, San Diego, Calif.) instead of anti-IgEantibodies or an IgE-specific allergen. Specific assays for assessingthe ability of a particular spiro 2,4-pyrimidinediamine compound of theinvention to inhibit ionophore-induced degranulation are provided in theExamples section.

As will be recognized by skilled artisans, compounds which exhibit ahigh degree of selectivity of FcεRI-mediated degranulation findparticular use, as such compounds selectively target the FcεRI cascadeand do not interfere with other degranulation mechanisms. Similarly,compounds which exhibit a high degree of selectivity of FcγRI-mediateddegranulation find particular use, as such compounds selectively targetthe FcγRI cascade and do not interfere with other degranulationmechanisms. Compounds which exhibit a high degree of selectivity aregenerally 10-fold or more selective for FcεRI- or FcγRI-mediateddegranulation over ionophore-induced degranulation, such asionomycin-induced degranulation.

Accordingly, the activity of spiro 2,4-pyrimidinediamine compounds mayalso be confirmed in biochemical or cellular assays of Syk kinaseactivity. Referring again to FIG. 2, in the FcεRI signaling cascade inmast and/or basophil cells, Syk kinase phosphorylates LAT andPLC-gammal, which leads to, among other things, degranulation. Any ofthese activities may be used to confirm the activity of spiro2,4-pyrimidinediamine compounds. In one embodiment, the activity isconfirmed by contacting an isolated Syk kinase, or an active fragmentthereof with a spiro 2,4-pyrimidinediamine compound in the presence of aSyk kinase substrate (e.g., a synthetic peptide or a protein that isknown to be phosphorylated by Syk in a signaling cascade) and assessingwhether the Syk kinase phosphorylated the substrate. Alternatively, theassay may be carried out with cells that express a Syk kinase. The cellsmay express the Syk kinase endogenously or they may be engineered toexpress a recombinant Syk kinase. The cells may optionally also expressthe Syk kinase substrate. Cells suitable for performing suchconfirmation assays, as well as methods of engineering suitable cellswill be apparent to those of skill in the art. Specific examples ofbiochemical and cellular assays suitable for confirming the activity ofspiro 2,4-pyrimidinediamine compounds are provided in the Examplessection.

Generally, compounds that are Syk kinase inhibitors will exhibit an IC₅₀with respect to a Syk kinase activity, such as the ability of Syk kinaseto phosphorylate a synthetic or endogenous substrate, in an in vitro orcellular assay in the range of about 20 μM or less. Skilled artisanswill appreciate that compounds that exhibit lower IC₅₀s, such as in therange of 10 μM, 1 μM, 100 nM, 10 nM, 1 nM, or even lower, areparticularly useful.

6.6 Uses and Compositions

As previously discussed, active compounds inhibit Fc receptor signalingcascades, especially those Fc receptors including a gamma homodimer,such as the FcεRI and/or FcγRI signaling cascades, that lead to, amongother things, the release and/or synthesis of chemical mediators fromcells, either via degranulation or other processes. As also discussed,the active compounds are also potent inhibitors of Syk kinase. As aconsequence of these activities, the active compounds of the inventionmay be used in a variety of in vitro, in vivo and ex vivo contexts toregulate or inhibit Syk kinase, signaling cascades in which Syk kinaseplays a role, Fc receptor signaling cascades, and the biologicalresponses effected by such signaling cascades. For example, in oneembodiment, the compounds may be used to inhibit Syk kinase, either invitro or in vivo, in virtually any cell type expressing Syk kinase. Theymay also be used to regulate signal transduction cascades in which Sykkinase plays a role. Such Syk-dependent signal transduction cascadesinclude, but are not limited to, the FcεRI, FcγRI, FcγRIII, BCR andintegrin signal transduction cascades. The compounds may also be used invitro or in vivo to regulate, and in particular inhibit, cellular orbiological responses effected by such Syk-dependent signal transductioncascades. Such cellular or biological responses include, but are notlimited to, respiratory burst, cellular adhesion, cellulardegranulation, cell spreading, cell migration, cell aggregation,phagocytosis, cytokine synthesis and release, cell maturation and Ca²⁺flux. Importantly, the compounds may be used to inhibit Syk kinase invivo as a therapeutic approach towards the treatment or prevention ofdiseases mediated, either wholly or in part, by a Syk kinase activity.Non-limiting examples of Syk kinase mediated diseases that may betreated or prevented with the compounds are those discussed in moredetail, below.

In another embodiment, the active compounds may be used to regulate orinhibit the Fc receptor signaling cascades and/or FcεRI- and/orFcγRI-mediated degranulation as a therapeutic approach towards thetreatment or prevention of diseases characterized by, caused by and/orassociated with the release or synthesis of chemical mediators of suchFc receptor signaling cascades or degranulation. Such treatments may beadministered to animals in veterinary contexts or to humans. Diseasesthat are characterized by, caused by or associated with such mediatorrelease, synthesis or degranulation, and that can therefore be treatedor prevented with the active compounds include, by way of example andnot limitation, atopy or anaphylactic hypersensitivity or allergicreactions, allergies (e.g., allergic conjunctivitis, allergic rhinitis,atopic asthma, atopic dermatitis and food allergies), low grade scarring(e.g., of scleroderma, increased fibrosis, keloids, post-surgical scars,pulmonary fibrosis, vascular spasms, migraine, reperfusion injury andpost myocardial infarction), diseases associated with tissue destruction(e.g., of COPD, cardiobronchitis and post myocardial infarction),diseases associated with tissue inflammation (e.g., irritable bowelsyndrome, spastic colon and inflammatory bowel disease), inflammationand scarring.

In addition to the myriad diseases discussed above, spiro2,4-pyrimidinediamine compounds described herein may also be useful forthe treatment or prevention of autoimmune diseases, as well as thevarious symptoms associated with such diseases. The types of autoimmunediseases that may be treated or prevented with the 2,4-pyrimidinediaminecompounds generally include those disorders involving tissue injury thatoccurs as a result of a humoral and/or cell-mediated response toimmunogens or antigens of endogenous and/or exogenous origin. Suchdiseases are frequently referred to as diseases involving thenonanaphylactic (i.e., Type II, Type III and/or Type IV)hypersensitivity reactions.

As discussed previously, Type I hypersensitivity reactions generallyresult from the release of pharmacologically active substances, such ashistamine, from mast and/or basophil cells following contact with aspecific exogenous antigen. As mentioned above, such Type I reactionsplay a role in numerous diseases, including allergic asthma, allergicrhinitis, etc.

Type II hypersensitivity reactions (also referred to as cytotoxic,cytolytic complement-dependent or cell-stimulating hypersensitivityreactions) result when immunoglobulins react with antigenic componentsof cells or tissue, or with an antigen or hapten that has becomeintimately coupled to cells or tissue. Diseases that are commonlyassociated with Type II hypersensitivity reactions include, but are notlimited, to autoimmune hemolytic anemia, erythroblastosis fetalis andGoodpasture's disease.

Type III hypersensitivity reactions, (also referred to as toxic complex,soluble complex, or immune complex hypersensitivity reactions) resultfrom the deposition of soluble circulating antigen-immunoglobulincomplexes in vessels or in tissues, with accompanying acute inflammatoryreactions at the site of immune complex deposition. Non-limitingexamples of prototypical Type III reaction diseases include the Arthusreaction, rheumatoid arthritis, serum sickness, systemic lupuserythematosis, certain types of glomerulonephritis, multiple sclerosisand bullous pemphingoid.

Type IV hypersensitivity reactions (frequently called cellular,cell-mediated, delayed, or tuberculin-type hypersensitivity reactions)are caused by sensitized T-lymphocytes which result from contact with aspecific antigen. Non-limiting examples of diseases cited as involvingType IV reactions are contact dermatitis and allograft rejection.

Autoimmune diseases associated with any of the above nonanaphylactichypersensitivity reactions may be treated or prevented with spiro2,4-pyrimidinediamine. In particular, the methods may be used to treator prevent those autoimmune diseases frequently characterized as singleorgan or single cell-type autoimmune disorders including, but notlimited to: Hashimoto's thyroiditis, autoimmune hemolytic anemia,autoimmune atrophic gastritis of pernicious anemia, autoimmuneencephalomyelitis, autoimmune orchitis, Goodpasture's disease,autoimmune thrombocytopenia, sympathetic ophthalmia, myasthenia gravis,Graves' disease, primary biliary cirrhosis, chronic aggressivehepatitis, ulcerative colitis and membranous glomerulopathy, as well asthose autoimmune diseases frequently characterized as involving systemicautoimmune disorder, which include but are not limited to: systemiclupus erythematosis, rheumatoid arthritis, Sjogren's syndrome, Reiter'ssyndrome, polymyositis-dermatomyositis, systemic sclerosis,polyarteritis nodosa, multiple sclerosis and bullous pemphigoid.

It will be appreciated by skilled artisans that many of the above-listedautoimmune diseases are associated with severe symptoms, theamelioration of which provides significant therapeutic benefit even ininstances where the underlying autoimmune disease may not beameliorated. Many of these symptoms, as well as their underlying diseasestates, result as a consequence of activating the FcγR signaling cascadein monocyte cells. As the 2,4-pyrimidinediamine compounds describedherein are potent inhibitors of such FcγR signaling in monocytes andother cells, the methods find use in the treatment and/or prevention ofmyriad adverse symptoms associated with the above-listed autoimmunediseases.

As a specific example, rheumatoid arthritis (RA) typically results inswelling, pain, loss of motion and tenderness of target jointsthroughout the body. RA is characterized by chronically inflamedsynovium that is densely crowded with lymphocytes. The synovialmembrane, which is typically one cell layer thick, becomes intenselycellular and assumes a form similar to lymphoid tissue, includingdendritic cells, T-, B- and NK cells, macrophages and clusters of plasmacells. This process, as well as a plethora of immunopathologicalmechanisms including the formation of antigen-immunoglobulin complexes,eventually result in destruction of the integrity of the joint,resulting in deformity, permanent loss of function and/or bone erosionat or near the joint. The methods may be used to treat or ameliorate anyone, several or all of these symptoms of RA. Thus, in the context of RA,the methods are considered to provide therapeutic benefit (discussedmore generally, infra) when a reduction or amelioration of any of thesymptoms commonly associated with RA is achieved, regardless of whetherthe treatment results in a concomitant treatment of the underlying RAand/or a reduction in the amount of circulating rheumatoid factor(“RF”).

As another specific example, systemic lupus erythematosis (“SLE”) istypically associated with symptoms such as fever, joint pain(arthralgias), arthritis, and serositis (pleurisy or pericarditis). Inthe context of SLE, the methods are considered to provide therapeuticbenefit when a reduction or amelioration of any of the symptoms commonlyassociated with SLE are achieved, regardless of whether the treatmentresults in a concomitant treatment of the underlying SLE.

As another specific example, multiple sclerosis (“MS”) cripples thepatient by disturbing visual acuity; stimulating double vision;disturbing motor functions affecting walking and use of the hands;producing bowel and bladder incontinence; spasticity; and sensorydeficits (touch, pain and temperature sensitivity). In the context ofMS, the methods are considered to provide therapeutic benefit when animprovement or a reduction in the progression of any one or more of thecrippling effects commonly associated with MS is achieved, regardless ofwhether the treatment results in a concomitant treatment of theunderlying MS.

When used to treat or prevent such diseases, the active compounds may beadministered singly, as mixtures of one or more active compounds or inmixture or combination with other agents useful for treating suchdiseases and/or the symptoms associated with such diseases. The activecompounds may also be administered in mixture or in combination withagents useful to treat other disorders or maladies, such as steroids,membrane stabilizers, 5LO inhibitors, leukotriene synthesis and receptorinhibitors, inhibitors of IgE isotype switching or IgE synthesis, IgGisotype switching or IgG synthesis, β-agonists, tryptase inhibitors,aspirin, COX inhibitors, methotrexate, anti-TNF drugs, retuxin, PD4inhibitors, p38 inhibitors, PDE4 inhibitors, and antihistamines, to namea few. The active compounds may be administered per se in the form ofprodrugs or as pharmaceutical compositions, comprising an activecompound or prodrug.

Pharmaceutical compositions comprising the active compounds describedherein (or prodrugs thereof) may be manufactured by means ofconventional mixing, dissolving, granulating, dragee-making levigating,emulsifying, encapsulating, entrapping or lyophilization processes. Thecompositions may be formulated in conventional manner using one or morephysiologically acceptable carriers, diluents, excipients or auxiliarieswhich facilitate processing of the active compounds into preparationswhich can be used pharmaceutically.

The active compound or prodrug may be formulated in the pharmaceuticalcompositions per se, or in the form of a hydrate, solvate, N-oxide orpharmaceutically acceptable salt, as previously described. Typically,such salts are more soluble in aqueous solutions than the correspondingfree acids and bases, but salts having lower solubility than thecorresponding free acids and bases may also be formed.

Pharmaceutical compositions of the invention may take a form suitablefor virtually any mode of administration, including, for example,topical, ocular, oral, buccal, systemic, nasal, injection, transdermal,rectal, vaginal, etc., or a form suitable for administration byinhalation or insufflation.

For topical administration, the active compound(s) or prodrug(s) may beformulated as solutions, gels, ointments, creams, suspensions, etc. asare well-known in the art.

Systemic formulations include those designed for administration byinjection, e.g., subcutaneous, intravenous, intramuscular, intrathecalor intraperitoneal injection, as well as those designed for transdermal,transmucosal oral or pulmonary administration.

Useful injectable preparations include sterile suspensions, solutions oremulsions of the active compound(s) in aqueous or oily vehicles. Thecompositions may also contain formulating agents, such as suspending,stabilizing and/or dispersing agent. The formulations for injection maybe presented in unit dosage form, e.g., in ampoules or in multidosecontainers, and may contain added preservatives.

Alternatively, the injectable formulation may be provided in powder formfor reconstitution with a suitable vehicle, including but not limited tosterile pyrogen free water, buffer, dextrose solution, etc., before use.To this end, the active compound(s) may be dried by any art-knowntechnique, such as lyophilization, and reconstituted prior to use.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants are knownin the art.

For oral administration, the pharmaceutical compositions may take theform of, for example, lozenges, tablets or capsules prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(e.g., magnesium stearate, talc or silica); disintegrants (e.g., potatostarch or sodium starch glycolate); or wetting agents (e.g., sodiumlauryl sulfate). The tablets may be coated by methods well known in theart with, for example, sugars, films or enteric coatings.

Liquid preparations for oral administration may take the form of, forexample, elixirs, solutions, syrups or suspensions, or they may bepresented as a dry product for constitution with water or other suitablevehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethylalcohol, Cremophore™ or fractionated vegetable oils); and preservatives(e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). Thepreparations may also contain buffer salts, preservatives, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound or prodrug, as is well known.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For rectal and vaginal routes of administration, the active compound(s)may be formulated as solutions (for retention enemas) suppositories orointments containing conventional suppository bases such as cocoa butteror other glycerides.

For nasal administration or administration by inhalation orinsufflation, the active compound(s) or prodrug(s) can be convenientlydelivered in the form of an aerosol spray from pressurized packs or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, fluorocarbons, carbon dioxide or othersuitable gas. In the case of a pressurized aerosol, the dosage unit maybe determined by providing a valve to deliver a metered amount. Capsulesand cartridges for use in an inhaler or insufflator (for examplecapsules and cartridges comprised of gelatin) may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

A specific example of an aqueous suspension formulation suitable fornasal administration using commercially-available nasal spray devicesincludes the following ingredients: active compound or prodrug (0.5-20mg/ml); benzalkonium chloride (0.1-0.2 mg/mL); polysorbate 80 (TWEEN®80; 0.5-5 mg/ml); carboxymethylcellulose sodium or microcrystallinecellulose (1-15 mg/ml); phenylethanol (1-4 mg/ml); and dextrose (20-50mg/ml). The pH of the final suspension can be adjusted to range fromabout pH5 to pH7, with a pH of about pH 5.5 being typical.

Another specific example of an aqueous suspension suitable foradministration of the compounds via inhalation, and in particular forsuch administration of a compound of the invention, contains 1-20 mg/mLof the compound or prodrug, 0.1-1% (v/v) Polysorbate 80 (TWEEN®80), 50mM citrate and/or 0.9% sodium chloride.

For ocular administration, the active compound(s) or prodrug(s) may beformulated as a solution, emulsion, suspension, etc. suitable foradministration to the eye. A variety of vehicles suitable foradministering compounds to the eye are known in the art. Specificnon-limiting examples are described in U.S. Pat. No. 6,261,547; U.S.Pat. No. 6,197,934; U.S. Pat. No. 6,056,950; U.S. Pat. No. 5,800,807;U.S. Pat. No. 5,776,445; U.S. Pat. No. 5,698,219; U.S. Pat. No.5,521,222; U.S. Pat. No. 5,403,841; U.S. Pat. No. 5,077,033; U.S. Pat.No. 4,882,150; and U.S. Pat. No. 4,738,851.

For prolonged delivery, the active compound(s) or prodrug(s) can beformulated as a depot preparation for administration by implantation orintramuscular injection. The active ingredient may be formulated withsuitable polymeric or hydrophobic materials (e.g., as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives, e.g., as a sparingly soluble salt. Alternatively,transdermal delivery systems manufactured as an adhesive disc or patchwhich slowly releases the active compound(s) for percutaneous absorptionmay be used. To this end, permeation enhancers may be used to facilitatetransdermal penetration of the active compound(s). Suitable transdermalpatches are described in for example, U.S. Pat. No. 5,407,713; U.S. Pat.No. 5,352,456; U.S. Pat. No. 5,332,213; U.S. Pat. No. 5,336,168; U.S.Pat. No. 5,290,561; U.S. Pat. No. 5,254,346; U.S. Pat. No. 5,164,189;U.S. Pat. No. 5,163,899; U.S. Pat. No. 5,088,977; U.S. Pat. No.5,087,240; U.S. Pat. No. 5,008,110; and U.S. Pat. No. 4,921,475.

Alternatively, other pharmaceutical delivery systems may be employed.Liposomes and emulsions are well-known examples of delivery vehiclesthat may be used to deliver active compound(s) or prodrug(s). Certainorganic solvents such as dimethylsulfoxide (DMSO) may also be employed,although usually at the cost of greater toxicity.

The pharmaceutical compositions may, if desired, be presented in a packor dispenser device which may contain one or more unit dosage formscontaining the active compound(s). The pack may, for example, comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.

6.7 Effective Dosages

The active compound(s) or prodrug(s), or compositions thereof, willgenerally be used in an amount effective to achieve the intended result,for example in an amount effective to treat or prevent the particulardisease being treated. The compound(s) may be administeredtherapeutically to achieve therapeutic benefit or prophylactically toachieve prophylactic benefit. By therapeutic benefit is meanteradication or amelioration of the underlying disorder being treatedand/or eradication or amelioration of one or more of the symptomsassociated with the underlying disorder such that the patient reports animprovement in feeling or condition, notwithstanding that the patientmay still be afflicted with the underlying disorder. For example,administration of a compound to a patient suffering from an allergyprovides therapeutic benefit not only when the underlying allergicresponse is eradicated or ameliorated, but also when the patient reportsa decrease in the severity or duration of the symptoms associated withthe allergy following exposure to the allergen. As another example,therapeutic benefit in the context of asthma includes an improvement inrespiration following the onset of an asthmatic attack, or a reductionin the frequency or severity of asthmatic episodes. Therapeutic benefitalso includes halting or slowing the progression of the disease,regardless of whether improvement is realized.

For prophylactic administration, the compound may be administered to apatient at risk of developing one of the previously described diseases.For example, if it is unknown whether a patient is allergic to aparticular drug, the compound may be administered prior toadministration of the drug to avoid or ameliorate an allergic responseto the drug. Alternatively, prophylactic administration may be appliedto avoid the onset of symptoms in a patient diagnosed with theunderlying disorder. For example, a compound may be administered to anallergy sufferer prior to expected exposure to the allergen. Compoundsmay also be administered prophylactically to healthy individuals who arerepeatedly exposed to agents known to one of the above-describedmaladies to prevent the onset of the disorder. For example, a compoundmay be administered to a healthy individual who is repeatedly exposed toan allergen known to induce allergies, such as latex, in an effort toprevent the individual from developing an allergy. Alternatively, acompound may be administered to a patient suffering from asthma prior topartaking in activities which trigger asthma attacks to lessen theseverity of, or avoid altogether, an asthmatic episode.

The amount of compound administered will depend upon a variety offactors, including, for example, the particular indication beingtreated, the mode of administration, whether the desired benefit isprophylactic or therapeutic, the severity of the indication beingtreated and the age and weight of the patient, the bioavailability ofthe particular active compound, etc. Determination of an effectivedosage is well within the capabilities of those skilled in the art.

Effective dosages may be estimated initially from in vitro assays. Forexample, an initial dosage for use in animals may be formulated toachieve a circulating blood or serum concentration of active compoundthat is at or above an IC₅₀ of the particular compound as measured in asin vitro assay, such as the in vitro CHMC or BMMC and other in vitroassays described in the Examples section. Calculating dosages to achievesuch circulating blood or serum concentrations taking into account thebioavailability of the particular compound is well within thecapabilities of skilled artisans. For guidance, the reader is referredto Fingl & Woodbury, “General Principles,” In. Goodman and Gilman's ThePharmaceutical Basis of Therapeutics, Chapter 1, pp. 1-46, latestedition, Pergamon Press, and the references cited therein.

Initial dosages can also be estimated from in vivo data, such as animalmodels.

Animal models useful for testing the efficacy of compounds to treat orprevent the various diseases described above are well-known in the art.Suitable animal models of hypersensitivity or allergic reactions aredescribed in Foster, 1995, Allergy 50(21Suppl):6-9, discussion 34-38 andTumas et al., 2001, J. Allergy Clin. Immunol. 107(6):1025-1033. Suitableanimal models of allergic rhinitis are described in Szelenyi et al.,2000, Arzneimittelforschung 50(11): 1037-42; Kawaguchi et al., 1994,Clin. Exp. Allergy 24(3):238-244 and Sugimoto et al., 2000,Immunopharmacology 48(1): 1-7. Suitable animal models of allergicconjunctivitis are described in Carreras et al., 1993, Br. J. Opthalmol.77(8):509-514; Saiga et al., 1992, Ophthalmic Res. 24(1):45-50; andKunert et al., 2001, Invest. Opthalmol. Vis. Sci. 42(11):2483-2489.Suitable animal models of systemic mastocytosis are described in O'Keefeet al., 1987, J. Vet. Intern. Med. 1(2):75-80 and Bean-Knudsen et al.,1989, Vet. Pathol. 26(1):90-92. Suitable animal models of hyper IgEsyndrome are described in Claman et al., 1990, Clin. Immunol.Immunopathol. 56(1):46-53. Suitable animal models of B-cell lymphoma aredescribed in Hough et al., 1998, Proc. Natl. Acad. Sci. USA95:13853-13858 and Hakim et al., 1996, J. Immunol. 157(12):5503-5511.Suitable animal models of atopic disorders such as atopic dermatitis,atopic eczema and atopic asthma are described in Chan et al., 2001, J.Invest. Dermatol. 117(4):977-983 and Suto et al., 1999, Int. Arch.Allergy Immunol. 120(Suppl 1):70-75. Ordinarily skilled artisans canroutinely adapt such information to determine dosages suitable for humanadministration. Additional suitable animal models are described in theExamples section.

Dosage amounts will typically be in the range of from about 0.0001 or0.001 or 0.01 mg/kg/day to about 100 mg/kg/day, but may be higher orlower, depending upon, among other factors, the activity of thecompound, its bioavailability, the mode of administration and variousfactors discussed above. Dosage amount and interval may be adjustedindividually to provide plasma levels of the compound(s) which aresufficient to maintain therapeutic or prophylactic effect. For example,the compounds may be administered once per week, several times per week(e.g., every other day), once per day or multiple times per day,depending upon, among other things, the mode of administration, thespecific indication being treated and the judgment of the prescribingphysician. In cases of local administration or selective uptake, such aslocal topical administration, the effective local concentration ofactive compound(s) may not be related to plasma concentration. Skilledartisans will be able to optimize effective local dosages without undueexperimentation.

Preferably, the compound(s) will provide therapeutic or prophylacticbenefit without causing substantial toxicity. Toxicity of thecompound(s) may be determined using standard pharmaceutical procedures.The dose ratio between toxic and therapeutic (or prophylactic) effect isthe therapeutic index. Compounds(s) that exhibit high therapeuticindices are preferred.

The invention having been described, the following examples are offeredby way of illustration and not limitation.

7. EXAMPLES 7.2 Spiro 2,4-Pyrimidinediamine Compounds 7.2.1Racemic-2-(2-t-Butyldimethylsiloxyethyl)-6-nitro-3-oxo-4H-benz[1,4]oxazine

To solution of 4.8 gracemic-2-(2-hydroxyethyl)-6-nitro-3-oxo-4H-benz[1,4]oxazine indimethylformamide (100 mL) and 3.4 mL diethylisopropylamine at 0° C. wasadded 4.7 g tert-butyldimethylsilylchloride. The reaction mixture wasstirred at 0° C. for 20 minutes, at room temperature for 3 hour,concentrated and the resulting residue was partitioned between sodiumbicarbonate solution and EtOAc. The aqueous phase was extracted withEtOAc and the combined organics washed with water, brine then dried overMgSO₄. The solvent was removed by in vacuo and the crude materialpurified by column chromatography (hexanes/EtOAc) to yield 1 g of thedesired productracemic-2-(2-t-butyldimethylsiloxyethyl)-6-nitro-3-oxo-4H-benz[1,4]oxazineMS (m/e): 353 (MH⁺).

7.2.2Racemic-2-2-t-Butyldimethylsiloxyethyl-6-nitro-3-oxo-4-p-methoxybenzyl-benz[1,4]oxazine

A solution of 450 mg ofracemic-2-(2-t-butyldimethylsiloxyethyl)-6-nitro-3-oxo-4-(p-methoxybenzyl)-benz[1,4]oxazinein 25 mL of 1% concentrated HCl in EtOH was stirred for 2 h. The solventwas removed by rotary evaporation to yield 370 mg of the desired productracemic-2-(2-Hydroxyethyl)-6-nitro-3-oxo-4-(p-methoxybenzyl)-benz[1,4]oxazine.MS (m/e): 357 (MH⁺).

7.2.3Racemic-2-2-Methanesulfonylethyl)-6-nitro-3-oxo-4-p-methoxybenzyl-benz[1,4]oxazine

To solution of 640 mgracemic-2-(2-hydroxyethyl)-6-nitro-3-oxo-4-(p-methoxybenzyl)-benz[1,4]oxazinein THF (30 mL) and 1 mL DIEA at 0° C. was added 330 uL MsCl. Thereaction mixture was stirred at 0° C. for 20 minutes, at roomtemperature for 3 hours and then concentrated. The resulting residue waspartitioned between bicarbonate solution and EtOAc. The aqueous phasewas extracted with EtOAc and the combined organics washed with water,brine then dried over MgSO₄. The solvent was removed in vacuo and thecrude material purified by column chromatography (hexanes/EtOAc) toyield 1 g of the title compound. MS (m/e): 437 (MH⁺).

7.2.4 2-2-Cyclopropyl)-6-nitro-3-oxo-4-p-methoxybenzyl)-benz[1,4]oxazine

To solution of 500 mgracemic-2-(2-Methanesulfonylethyl)-6-nitro-3-oxo-4-(p-methoxybenzyl)-benz[1,4]oxazinein THF (30 mL) at −15° C. was slowly added (dropwise) 2.5 mL of a 1.0 Mlithiumhexamethylsilylazide solution and the reaction mixture wasstirred at −15° C. for 3 hour. The reaction mixture was concentrated andthe resulting residue was partitioned between bicarbonate solution andEtOAc. The aqueous phase was extracted with EtOAc and the combinedorganics washed with water, brine then dried over MgSO4. The solvent wasremoved in vacuo and the crude material purified by columnchromatography (hexanes/EtOAc) to yield 150 mg of the desired product2-2-cyclopropyl)-6-nitro-3-oxo-4-(p-methoxybenzyl)-benz[1,4]oxazine MS(m/e): 341 (MH+).

7.2.5 2-(2-Cyclopropyl)-6-nitro-3-oxo-4H-benz[1,4]oxazine

To solution of 400 mg2-(2-cyclopropyl)-6-nitro-3-oxo-4-(p-methoxybenzyl)-benz[1,4]oxazine inacetonitrile/water (50 mL) was added 2.5 eq of ceric ammonium nitrate.The reaction mixture was stirred at ambient temperature for 18 hour,filtered, concentrated and the resulting residue partitioned betweenbicarbonate solution and EtOAc. The aqueous phase was extracted withEtOAc and the combined organics washed with water, brine and then driedover MgSO₄. The solvent was removed in vacuo and the crude materialpurified by column chromatography (hexanes/EtOAc) to yield 250 mg of thedesired product 2-(2-cyclopropyl)-6-nitro-3-oxo-4H-benz[1,4]oxazine. MS(m/e): 341 (MH⁺).

7.2.6 6-Amino-2-(2-cyclopropyl)-3-oxo-4H-benz[1,4]oxazine

To a solution of 200 mg of2-(2-cyclopropyl)-6-nitro-3-oxo-4H-benz[1,4]oxazine (1 g) in EtOH/water(20 mL; 2:1 v/v) was added 5 eq of iron and 5 eq of ammonium chlorideand the reaction was heated at 90° C. for 4 h. The reaction mixture wascooled to room temperature, diluted with dichloromethane and filtered.The layers were separated, the aqueous was extracted once withdichloromethane, the combined organics layers were dried over magnesiumsulfate, the solution filtered and the filtrate evaporated in vacuo togive the desired product 6-amino-2-(2-cyclopropyl)-3-oxo-4H-benz [1,4].MS (m/e): 191 (MH⁺).

7.2.7 Pyrogallol 1-Benzenesulphonate 2,3-Dimethyl Ether

To solution of 10 mL 2,3-dimethoxyphenol and 13.3 mL TEA in 150 mL DCMat 0° C. was added 11.25 mL benzenesulphonyl chloride slowly dropwisewith stirring. The reaction mixture was allowed to warm to roomtemperature and stirred overnight. The reaction mixture was filtered anthe volume was reduce by rotary evaporation. The DCM solution was washedwith dilute HCl solution, 50% saturated bicarbonate solution then brine,dried with magnesium sulfate, filtered and the solvent evaporated toyield 11 g of the desired product pyrogallol 1-benzenesulphonate2,3-dimethyl ether. MS (m/e): 295 (MH⁺).

7.2.8 5-Nitro Pyrogallol 1-Benzenesulphonate 2,3-Dimethyl Ether

To solution of 6.26 g of pyrogallol 1-benzenesulphonate 2,3-dimethylether in 60 mL of glacial acetic acid at 0° C. was added 12 mL of fumingnitric acid. The reaction mixture was allowed to warm to roomtemperature and stirred overnight and then added to 400 mL of ice water.The solution was neutralized with solid sodium bicarbonate and extractedwith ethyl acetate. The combined organics were washed with saturatedbicarbonate solution, brine, dried over magnesium sulfate, filtered andevaporated and the resulting residue was purified by silica gel columnchromatography (hexanes/EtOAc 85:15) to yield 6 g of the desired product5-nitro pyrogallol 1-benzenesulphonate 2,3-dimethyl ether. MS (m/e): 340(MH⁺).

7.2.9 2,3-Dimethoxy-5-nitrophenol

A suspension of 6 g of (±)-5-nitro pyrogallol 1-benzenesulphonate2,3-dimethyl ether in 60 mL of methanol and 36 mL 20% by weight KOHaqueous solution was heated at 50 degrees Celcius for 30 minutes withstirring then cooled, diluted with 350 mL D.I. water and acidified withexcess conc. HCl to yield a precipitate that was collected by suctionfiltration and dried to yield 4.6 g of the desired product2,3-dimethoxy-5-nitrophenol. MS (m/e): 200 (MH⁺).

7.2.10 4,5-Dimethoxy-3-hydroxyaniline

A solution of 1.1 g of 2,3-dimethoxy-5-nitrophenol in 45 mL dry methanolwith 200 mg of 10% Pd/C (Degussa) was hydrogenated under a hydrogenballoon overnight. The reaction was filtered through a bed of celite andevaporated and the residue was chromatographed over silica gel (ethylacetate/hexanes 3:2 gradient to 100% ethyl acetate). The appropriatefractions were evaporated to dryness to yield an oil that was taken upin methanol to which methanolic HCl solution was added and the solventevaporated to yield 1.0 g of the desired product4,5-dimethoxy-3-hydroxyaniline hydrochloride. MS (m/e): 170 (MH⁺).

7.2.112-Chloro-N-4-[2-(2-cyclopropyl)-3-oxo-4H-benz[1,4]oxazin-6-yl]-5-fluoro-4-pyrimidineamine

A mixture of 325 mg of 2,4-dichloro-5-fluoropyrimidine and 124 mg of6-amino-2-(2-cyclopropyl)-3-oxo-4H-benz[1,4]oxazine in 20 mL 1:1methanol/water was heated overnight at 80° C. and upon cooling wasdiluted with 1 N HCl solution. The precipitate was collected by suctionfiltration, dried, triturated with hexanes and dried again to yield 170mg of the desired product2-chloro-N-4-[2-(2-cyclopropyl)-3-oxo-4H-benz[1,4]oxazin-6-yl]-5-fluoro-4-pyrimidineamine¹H NMR (DMSO-d₆): δ 8.24 (d, 1H), 7.22 (d, 1H), 7.19 (dd, 1H), 6.83 (d,2H), 1.20 (m, 2H); LCMS: retention time 11.30 min; purity 94%; MS (m/e):321 (MH⁺).

7.2.12N4-[2-(2-Cyclopropyl)-3-oxo-4H-benz[1,4]oxazin-6-yl]-5-fluoro-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine(Compound 1)

A mixture of 40 mg of2-Chloro-N4-[2-(2-cyclopropyl)-3-oxo-4H-benz[1,4]oxazin-6-yl]-5-fluoro-4-pyrimidineamineand 48 mg of 3-(N-methylamino)carbonylmethyleneoxyaniline in 700 uL EtOHwas heated in the microwave at 180° C. for 4200 seconds. The precipitateformed was collected by suction filtration, dried, suspended in a dilutebicarbonate solution, sonicated, recollected by suction filtration anddried to yield 25 mg of the desired productN4-[2-(2-Cyclopropyl)-3-oxo-4H-benz[1,4]oxazin-6-yl]-5-fluoro-N2-[3-(N-methylamino)carbonylmethylene-oxyphenyl]-2,4-pyrimidinediamine¹H NMR (DMSO-d₆): δ 8.08 (d, 1H), 7.92 (m, 1H), 7.32 (m, 4H), 7.04 (t,1H), 6.81 (d, 1H), 6.43 (dd, 1H), 4.33 (s, 2H), 2.62 (d, 3H), 1.19 (m,4H); LCMS: retention time 9.35 min; purity 96%; MS (m/e): 465 (MH⁺).

7.2.13N4-[2-(2-Cyclopropyl)-3-oxo-4H-benz[1,4]oxazin-6-yl]-5-fluoro-N2-(indazol-6-yl)-2,4-pyrimidinediamine(Compound 2)

In like manner to the preparation ofN4-[2-(2-cyclopropyl)-3-oxo-4H-benz[1,4]oxazin-6-yl]-5-fluoro-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine,2-chloro-N4-[2-(2-cyclopropyl)-3-oxo-4H-benz[1,4]oxazin-6-yl]-5-fluoro-4-pyrimidineamineand 6-aminoindazole were reacted to yield the desired productN4-[2-(2-cyclopropyl)-3-oxo-4H-benz[1,4]oxazin-6-yl]-5-fluoro-N2-(indazol-6-yl)-2,4-pyrimidinediamine.¹H NMR (DMSO-d₆): δ 8.06 (m, 2H), 7.82 (s, 1H), 7.53 (d, 4H), 7.39 (dd,1H), 7.22 (m, 2H), 6.83 (d, 1H), 1.19 (m, 4H); LCMS: retention time 9.29min; purity 90%; MS (m/e): 418 (MH⁺).

7.2.14 N4-[2-(2-Cyclopropyl)-3-oxo-4H-benz[1,4]oxazin-6-yl]-5-fluoro-N2-(3-hydroxyphenyl)-2,4-pyrimidinediamine(Compound 3)

In like manner to the preparation ofN4-[2-(2-cyclopropyl)-3-oxo-4H-benz[1,4]oxazin-6-yl]-5-fluoro-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine,2-chloro-N4-[2-(2-cyclopropyl)-3-oxo-4H-benz[1,4]oxazin-6-yl]-5-fluoro-4-pyrimidineamineand 3-hydroxyaniline were reacted to yield the desired productN4-[2-(2-cyclopropyl)-3-oxo-4H-benz[1,4]oxazin-6-yl]-5-fluoro-N2-(3-hydroxyphenyl)-2,4-pyrimidinediamine.¹H NMR (DMSO-d₆): δ 8.09 (d, 1H), 7.23 (m, 2H), 7.01 (m, 3H), 6.82 (d,1H), 6.39 (m, 1H), 1.20 (m, 4H); LCMS: retention time 9.30 min; purity96%; MS (m/e): 394 (MH⁺).

7.2.15N4-[2-(2-Cyclopropyl)-3-oxo-4H-benz[1,4]oxazin-6-yl]-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine(Compound 4)

In like manner to the preparation ofN4-[2-(2-cyclopropyl)-3-oxo-4H-benz[1,4]oxazin-6-yl]-5-fluoro-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine,2-chloro-N4-[2-(2-cyclopropyl)-3-oxo-4H-benz[1,4]oxazin-6-yl]-5-fluoro-4-pyrimidineamineand 3,4,5-trimethoxyaniline were reacted to yield the desired productN4-[2-(2-cyclopropyl)-3-oxo-4H-benz[1,4]oxazin-6-yl]-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine.¹H NMR (DMSO-d₆): δ 8.12 (d, 1H), 7.21 (m, 2H), 6.87 (s, 2H), 6.77 (d,1H), 3.58 (s, 6H), 3.56 (S, 3H), 1.20 (m, 4H); LCMS: retention time10.25 min; purity 96.5%; MS (m/e): 468 (MH⁺).

7.2.16 N4-[2-(2-Cyclopropyl)-3-oxo-4H-benz[1,4]oxazin-6-yl]-5-fluoro-N2-(3,4-dimethoxy-5-hydroxyphenyl)-2,4-pyrimidinediamine(Compound 5)

In like manner to the preparation ofN4-[2-(2-cyclopropyl)-3-oxo-4H-benz[1,4]oxazin-6-yl]-5-fluoro-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine,2-chloro-N4-[2-(2-cyclopropyl)-3-oxo-4H-benz[1,4]oxazin-6-yl]-5-fluoro-4-pyrimidineamineand, 5-dimethoxy-3-hydroxyaniline hydrochloride were reacted to yieldthe desired productN4-[2-(2-cyclopropyl)-3-oxo-4H-benz[1,4]oxazin-6-yl]-5-fluoro-N2-(3-hydroxy-4,5-trimethoxyphenyl)-2,4-pyrimidinediamine.¹H NMR (DMSO-d₆): δ 8.22 (s, 1H), 8.02 (d, 2H), 7.33 (m, 2H), 6.83 (m,3H), 3.56 (S, 6H), 1.20 (m, 4H); purity 99%; MS (m/e): 454 (MH⁺).

7.3 Assays for Determining Whether Spiro 2,4-Pyrimidinediamine CompoundsInhibit FcεRI Receptor-Mediated Degranulation

The ability of spiro 2,4-pyrimidinediamine compounds to inhibitIgE-induced degranulation may be demonstrated in a variety of cellularassays with cultured human mast cells (CHMC) and/or mouse bone marrowderived cells (BMMC). Inhibition of degranulation is measured at bothlow and high cell density by quantifying the release of the granulespecific factors tryptase, histamine and hexosaminidase. Inhibition ofrelease and/or synthesis of lipid mediators is assessed by measuring therelease of leukotriene LTC4 and inhibition of release and/or synthesisof cytokines was monitored by quantifying TNFα, IL-6 and IL-13. Tryptaseand hexosaminidase are quantified using fluorogenic substrates asdescribed in their respective examples. Histamine, TNFα, IL-6, IL-13 andLTC4 are quantified using the following commercial ELISA kits: histamine(Immunotech #2015, Beckman Coulter), TNFα (Biosource #KHC3011), IL-6(Biosource #KMC0061), IL-13 (Biosource #KHC0132) and LTC4 (CaymanChemical #520211). The protocols of the various assays are providedbelow.

7.3.1 Culturing of Human Mast and Basophil Cells

Human mast and basophil cells are cultured from CD34-negative progenitorcells as described below (see also the methods described in copendingU.S. application Ser. No. 10/053,355, filed Nov. 8, 2001).

7.3.2 Preparation of STEMPRO-34 Complete Medium

To prepare STEMPRO-34 complete medium (“CM”), 250 mL STEMPRO-34′ serumfree medium (“SFM”; GibcoBRL, Catalog No. 10640) is added to a filterflask. To this is added 13 mL STEMPRO-34 Nutrient Supplement (“NS”;GibcoBRL, Catalog No. 10641) (prepared as described in more detail,below). The NS container is rinsed with approximately 10 mL SFM and therinse is added to the filter flask. Following addition of 5 mLL-glutamine (200 mM; Mediatech, Catalog No. MT 25-005-CJ and 5 mL 100×penicillin/streptomycin (“pen-strep”; HyClone, Catalog No. SV30010), thevolume is brought to 500 mL with SFM and the solution is filtered.

The most variable aspect of preparing the CM is the method by which theNS is thawed and mixed prior to addition to the SFM. The NS should bethawed in a 37° C. water bath and is swirled, not vortexed or shaken,until it is completely in solution. While swirling, take note whetherthere are any lipids that are not yet in solution. If lipids are presentand the NS is not uniform in appearance, return it to the water bath andrepeat the swirling process until it is uniform in appearance. Sometimesthis component goes into solution immediately, sometimes after a coupleof swirling cycles, and sometimes not at all. If, after a couple ofhours, the NS is still not in solution, discard it and thaw a freshunit. NS that appears non-uniform after thaw should not be used.

7.3.3 Expansion of CD34+ Cells

A starting population of CD34-positive (CD34⁺) cells of relatively smallnumber (1−5×10⁶ cells) is expanded to a relatively large number ofCD34-negative progenitor cells (about 2−4×10⁹ cells) using the culturemedia and methods described below. The CD34+ cells (from a single donor)are obtained from Allcells (Berkeley, Calif.). Because there is a degreeof variation in the quality and number of CD34+ cells that Allcellstypically provides, the newly delivered cells are transferred to a 15 mLconical tube and brought up to 10 mL in CM prior to use.

On day 0, a cell count is performed on the viable (phase-bright) cellsand the cells were spun at 1200 rpm to pellet. The cells are resuspendedto a density of 275,000 cells/mL with CM containing 200 ng/mLrecombinant human Stem Cell Factor (“SCF”; Peprotech, Catalog No.300-07) and 20 ng/mL human flt-3 ligand (Peprotech, Catalog No. 300-19)(“CM/SCF/flt-3 medium”). On about day 4 or 5, the density of the cultureis checked by performing a cell count and the culture is diluted to adensity of 275,000 cells/mL with fresh CM/SCF/flt-3 medium. On about day7, the culture is transferred to a sterile tube and a cell count isperformed. The cells are spun at 1200 rpm and resuspended to a densityof 275,000 cells/mL with fresh CM/SCF/flt-3 medium.

This cycle is repeated, starting from day 0, a total of 3-5 times overthe expansion period.

When the culture is large and being maintained in multiple flasks and isto be resuspended, the contents of all of the flasks are combined into asingle container prior to performing a cell count. This ensures that anaccurate cell count is achieved and provides for a degree of uniformityof treatment for the entire population. Each flask is checked separatelyfor contamination under the microscope prior to combining to preventcontamination of the entire population.

Between days 17-24, the culture can begin to go into decline (i.e.,approximately 5-10% of the total number of cells die) and fail to expandas rapidly as before. The cells are then monitored on a daily basisduring this time, as complete failure of the culture can take place inas little as 24 hours. Once the decline has begun, the cells arecounted, spun down at 850 rpm for 15 minutes, and resuspended at adensity of 350,000 cells/mL in CMNSCF/flt-3 medium to induce one or twomore divisions out of the culture. The cells are monitored daily toavoid failure of the culture.

When greater than 15% cell death is evident in the progenitor cellculture and some debris is present in the culture, the CD34-negativeprogenitor cells are ready to be differentiated.

7.3.4 Differentiation of CD34-Negative Progenitor Cells into MucosalMast Cells

A second phase is performed to convert the expanded CD34-negativeprogenitor cells into differentiated mucosal mast cells. These mucosalcultured human mast cells (“CHMC”) are derived from CD34+ cells isolatedfrom umbilical cord blood and treated to form a proliferated populationof CD34-negative progenitor cells, as described above. To produce theCD43-negative progenitor cells, the resuspension cycle for the cultureis the same as that described above, except that the culture is seededat a density of 425,000 cells/mL and 15% additional media is added onabout day four or five without performing a cell count. Also, thecytokine composition of the medium is modified such that it containedSCF (200 ng/mL) and recombinant human IL-6 (200 ng/mL; Peprotech,Catalog No. 200-06 reconstituted to 100 ug/mL in sterile 10 mM aceticacid) (“CM/SCF/IL-6 medium”).

Phases I and II together span approximately 5 weeks. Some death anddebris in the culture is evident during weeks 1-3 and there is a periodduring weeks 2-5 during which a small percentage of the culture is nolonger in suspension, but is instead attached to the surface of theculture vessel.

As during Phase I, when the culture is to be resuspended on day seven ofeach cycle, the contents of all flasks are combined into a singlecontainer prior to performing a cell count to ensure uniformity of theentire population. Each flask is checked separately for contaminationunder the microscope prior to combining to prevent contamination of theentire population.

When the flasks are combined, approximately 75% of the volume istransferred to the communal container, leaving behind about 10 mL or soin the flask. The flask containing the remaining volume is rappedsharply and laterally to dislodge the attached cells. The rapping isrepeated at a right angle to the first rap to completely dislodge thecells.

The flask is leaned at a 45 degree angle for a couple of minutes beforethe remaining volume is transferred to the counting vessel. The cellswere spun at 950 rpm for 15 min prior to seeding at 35-50 mL per flask(at a density of 425,000 cells/mL).

7.3.5 Differentiation of CD34-Negative Progenitor Cells into ConnectiveTissue-Type Mast Cells

A proliferated population of CD34-negative progenitor cells is preparedas above and treated to form a tryptase/chymase positive (connectivetissue) phenotype. The methods are performed as described above formucosal mast cells, but with the substitution of IL-4 for IL-6 in theculture medium. The cells obtained are typical of connective tissue mastcells.

7.3.6 Differentiation of CD34-Negative Progenitor Cells into BasophilCells

A proliferated population of CD34-negative progenitor cells is preparedas described in Section 7.2.1.3, above and used to form a proliferatedpopulation of basophil cells. The CD34-negative cells are treated asdescribed for mucosal mast cells, but with the substitution of IL-3 (at20-50 ng/mL) for IL-6 in the culture medium.

7.3.7 CHMC Low Cell Density IgE Activation Tryptase and LTC4 Assays

To duplicate 96-well U-bottom plates (Costar 3799) add 65 ul of compounddilutions or control samples that have been prepared in MT [137 mM NaCl,2.7 mM KCl, 1.8 mM CaCl₂, 1.0 mM MgCl₂, 5.6 mM Glucose, 20 mM Hepes (pH7.4), 0.1% Bovine Serum Albumin, (Sigma A4503)] containing 2% MeOH and1% DMSO. Pellet CHMC cells (980 rpm, 10 min) and resuspend in pre-warmedMT. Add 65 ul of cells to each 96-well plate. Depending on thedegranulation activity for each particular CHMC donor, load 1000-1500cells/well. Mix four times followed by a 1 hr incubation at 37° C.During the 1 hr incubation, prepare 6× anti-IgE solution [rabbitanti-human IgE (1 mg/ml, Bethyl Laboratories A80-109A) diluted 1:167 inMT buffer]. Stimulate cells by adding 25 ul of 6× anti-IgE solution tothe appropriate plates. Add 25 ul MT to un-stimulated control wells. Mixtwice following addition of the anti-IgE. Incubate at 37° C. for 30minutes. During the 30 minute incubation, dilute the 20 mM tryptasesubstrate stock solution [(Z-Ala-Lys-Arg-AMC-2TFA; Enzyme SystemsProducts, #AMC-246)] 1:2000 in tryptase assay buffer [0.1 M Hepes (pH7.5), 10% w/v Glycerol, 10 uM Heparin (Sigma H-4898) 0.01% NaN₃]. Spinplates at 1000 rpm for 10 min to pellet cells. Transfer 25 ul ofsupernatant to a 96-well black bottom plate and add 100 ul of freshlydiluted tryptase substrate solution to each well. Incubate plates atroom temperature for 30 min. Read the optical density of the plates at355 nm/460 nm on a spectrophotometric plate reader.

Leukotriene C4 (LTC4) is also quantified using an ELISA kit onappropriately diluted supernatant samples (determined empirically foreach donor cell population so that the sample measurement falls withinthe standard curve) following the supplier's instructions.

7.3.8 CHMC High Cell Density IgE Activation Degranulation (Tryptase,Histamine), Leukotriene (LTC4), and Cytokine (TNFalpha, IL-13) Assays

Cultured human mast cells (CHMC) are sensitized for 5 days with IL-4 (20ng/ml), SCF (200 ng/ml), IL-6 (200 ng/ml), and Human IgE (CP 1035K fromCortx Biochem, 100-500 ng/ml depending on generation) in CM medium.After sensitizing, cells are counted, pelleted (1000 rpm, 5-10 minutes),and resuspended at 1−2×10⁶ cells/ml in MT buffer. Add 100 ul of cellsuspension to each well and 100 ul of compound dilutions. The finalvehicle concentration is 0.5% DMSO. Incubate at 37 C (5% CO2) for 1hour. After 1 hour of compound treatment, stimulate cells with 6×anti-IgE. Mix wells with the cells and allow plates to incubate at 37 C(5% CO₂) for one hour. After 1 hour incubation, pellet cells (10minutes, 1000 RPM) and collect 200 ul per well of the supernatant, beingcareful not to disturb pellet. Place the supernatant plate on ice.During the 7-hour step (see next) perform tryptase assay on supernatantthat had been diluted 1:500. Resuspend cell pellet in 240 ul of CM mediacontaining 0.5% DMSO and corresponding concentration of compound.Incubate CHMC cells for 7 hours at 37° C. (5% CO₂). After incubation,pellet cells (1000 RPM, 10 minutes) and collect 225 ul per well andplace in −80° C. until ready to perform ELISAS. ELISAS are performed onappropriately diluted samples (determined empirically for each donorcell population so that the sample measurement falls within the standardcurve) following the supplier's instructions.

7.4 The 2,4-Pyrimidinediamine Compounds of the Invention May SelectivelyInhibit the Upstream IgE Receptor Cascade

The spiro 2,4-pyrimidinediamine compounds may be tested in cellularassays for ionomycin-induced degranulation, as described below, todetermine if they are blocking or inhibiting the early IgE receptorsignal transduction cascade.

7.4.1 CHMC Low Cell Density Ionomycin Activation Tryptase Assay

Assays for ionomycin-induced mast cell degranulation are carried out asdescribed for the CHMC Low Density IgE Activation assays (Section 7.2.2,supra), with the exception that during the 1 hour incubation, 6×ionomycin solution [5 mM ionomycin (Sigma 1-0634) in MeOH (stock)diluted 1:416.7 in MT buffer (2 M final)] is prepared and cells arestimulated by adding 25 μl of the 6× ionomycin solution to theappropriate plates.

7.5 The Spiro 2,4-Pyrimidinediamine Compounds Inhibit the IgE ReceptorSignalling Cascade

Compounds 1-5 were tested for their ability to inhibit the IgE receptorsignaling cascade in an 8-point assay with CHMC cells. For the assay theamount of tryptase release was measured. All five compounds exhibitedIC₅₀'s of less than 100 nM.

7.6 2,4-Pyrimidinediamine Compounds Inhibit Syk Kinase in BiochemicalAssays

The spiro 2,4-pyrimidinediamine compounds can be tested for the abilityto inhibit Syk kinase catalyzed phosphorylation of a peptide substratein a biochemical fluorescence polarization assay with isolated Sykkinase. In this experiment, compounds are diluted to 1% DMSO in kinasebuffer (20 mM HEPES, pH 7.4, 5 mM MgC₂, 2 mM MnCl₂, 1 mM DTT, 0.1 mg/mLacetylated Bovine Gamma Globulin). Compounds in 1% DMSO (0.2% DMSOfinal) are mixed with ATP/substrate solution at room temperature. Sykkinase (Upstate, Lake Placid N.Y.) is added to a final reaction volumeof 20 uL, and the reaction is incubated for 30 minutes at roomtemperature. Final enzyme reaction conditions are 20 mM HEPES, pH 7.4, 5mM MgCl₂, 2 mM MnCl₂, 1 mM DTT, 0.1 mg/mL acetylated Bovine GammaGlobulin, 0.125 ng Syk, 4 uM ATP, 2.5 uM peptide substrate(biotin-EQEDEPEGDYEEVLE-CONH2, SynPep Corporation). EDTA (10 mMfinal)/anti-phosphotyrosine antibody (1× final)/fluorescentphosphopeptide tracer (0.5× final) is added in FP Dilution Buffer tostop the reaction for a total volume of 40 uL according tomanufacturer's instructions (PanVera Corporation). The plate isincubated for 30 minutes in the dark at room temperature. Plates areread on a Polarion fluorescence polarization plate reader (Tecan). Datawas converted to an amount of phosphopeptide present using a calibrationcurve generated by competition with the phosphopeptide competitorprovided in the Tyrosine Kinase Assay Kit, Green (PanVera Corporation).

Compounds 1-4 were tested in this assay. All compounds exhibited an IC₅₀of Syk inhibition of less than 125 nM.

7.7 The In Vivo Efficacy of Compounds Towards Autoimmune Diseases May beDemonstrated in a Mouse Model of Collagen Antibody-Induced Arthritis(CAIA) 7.7.1 Model

Collagen-induced arthritis (CIA) in rodents is frequently used as one ofthe experimental models for IC-mediated tissue injury. Administration oftype II collagen into mice or rats results in an immune reaction thatcharacteristically involves inflammatory destruction of cartilage andbone of the distal joints with concomitant swelling of surroundingtissues. CIA is commonly used to evaluate compounds that might be ofpotential use as drugs for treatment of rheumatoid arthritis and otherchronic inflammatory conditions.

In recent years, a new technique emerged in CIA modeling, in which theanti-type II collagen antibodies are applied to induce anantibody-mediated CIA. The advantages of the method are: Short time forinduction of disease (developing within 24-48 hrs after an intravenous(IV) injection of antibodies); arthritis is inducible in bothCIA-susceptible and CIA-resistant mouse strains; and the procedure isideal for rapid screening of anti-inflammatory therapeutic agents.

Arthrogen-CIA® Arthritis-inducing Monoclonal Antibody Cocktail (ChemiconInternational Inc.) is administered intravenously to Balb/c mice (2mg/mouse) on Day 0. Forty-eight hours later, 100 μl of LPS (25 μg) isinjected intraperitoneally. On Day 4, toes may appear swollen. By Day 5,one or two paws (particular the hind legs) begin to appear red andswollen. On Day 6, and thereafter, red and swollen paws will remain forat least 1-2 weeks. During the study, the clinical signs of inflammationare scored to evaluate the intensity of edema in the paws. The severityof arthritis is recorded as the sum score of both hind paws for eachanimal (possible maximum score of 8). The degree of inflammation withinvolved paws is evaluated by measurement of diameter of the paws. Bodyweight changes are monitored.

Animals can be treated at the time of induction of arthritis, beginningon Day 0. Test compounds and control compounds can be administered oncea day (q.d.) or twice a day (b.i.d.), via per os (PO), depending onpreviously established PK profiles.

At the end of the study (1-2 weeks after induction of arthritis), miceare euthanized and the paws are transected at the distal tibia using aguillotine and weighed. The mean±standard error of the mean (SEM) foreach group is determined each day from individual animal clinicalscores, and hind paw weights for each experimental group are calculatedand recorded at study termination. Histopathological evaluation of pawsare obtained.

7.7.2 Results

Reduced inflammation and swelling should be evident in animals treatedwith compounds described herein, and the arthritis would progress moreslowly. Treatment with compounds should (b.i.d.) significantly reduceclinical arthritis compared with animals treated with vehicle only.

7.8 The Compounds can be Effective in Rat Collagen-Induced Arthritis

The in vivo efficacy of compounds described herein towards autoimmunediseases can be demonstrated in a rat model of collagen-inducedarthritis (CIA).

7.8.1 Model Description

Rheumatoid arthritis (RA) is characterized by chronic joint inflammationeventually leading to irreversible cartilage destruction. IgG-containingIC are abundant in the synovial tissue of patients with RA. While it isstill debated what role these complexes play in the etiology andpathology of the disease, IC communicate with the hematopoetic cells viathe FcγR.

CIA is a widely accepted animal model of RA that results in chronicinflammatory synovitis characterized by pannus formation and jointdegradation. In this model, intradermal immunization with native type IIcollagen, emulsified with incomplete Freund's adjuvant, results in aninflammatory polyarthritis within 10 or 11 days and subsequent jointdestruction in 3 to 4 weeks.

7.8.2 Study Protocol

Syngeneic LOU rats are immunized on Day 0 with native chicken CII/IFA(performed at UCLA; E. Brahn, Principal Investigator). Beginning on theday of arthritis onset (Day 10), a total of 59 rats are treated witheither a vehicle control or a compound described herein at one of fourdose levels (1, 3, 10, and 30 mg/kg, q.d. by p.o. gavage).

7.8.3 Results

Hind limbs are scored daily for clinical arthritis severity using astandardized method based on the degree of joint inflammation. Highresolution digital radiographs of hind limbs can be obtained at theconclusion of the study (Day 28). These limbs can also be analyzed forhistopathologic changes. IgG antibodies to native CII can be measured inquadruplicate by ELISA.

Although the foregoing invention has been described in some detail tofacilitate understanding, it will be apparent that certain changes andmodifications may be practiced within the scope of the appended claims.Accordingly, the described embodiments are to be considered asillustrative and not restrictive, and the invention is not to be limitedto the details given herein, but may be modified within the scope andequivalents of the appended claims.

All literature and patent references cited throughout the applicationare incorporated by reference into the application for all purposes intheir entirety.

1.-29. (canceled)
 30. A method of treating or preventing an autoimmunedisease and/or one or more symptoms associated therewith, comprising thestep of administering to a subject suffering from an autoimmune diseaseor at risk of developing an autoimmune disease an effective amount of a2,4-pyrimidinediamine compound according to structural formula (VI):

or a salt or N-oxide thereof, wherein: R² is selected from the groupconsisting of (C1-C6) alkyl optionally substituted with one or more ofthe same or different R⁸ groups, 3-8 membered cycloheteroalkyloptionally substituted with one or more of the same or different R⁸groups, (C5-C15) aryl optionally substituted with one or more of thesame or different R⁸ groups, and 5-15 membered heteroaryl optionallysubstituted with one or more of the same or different R⁸ groups; each Wis, independently of the other, —CR³¹R³¹—: X is selected from the groupconsisting of —N— and —CH—; Y and Z are each, independently of oneanother, selected from the group consisting of —O—, —S—, —SO—, —SO₂—,—SONR³⁶—, —NH—, and —NR³⁵; R⁵ is selected from the group consisting ofhydrogen, —OR^(d), —SR^(d), (C1-C3) haloalkyloxy, (C1-C3)perhaloalkyloxy, —NR^(c)R^(c), halogen, (C1-C3) haloalkyl, (C1-C3)perhaloalkyl, —CN, —NC, —OCN, —SCN, —NO, —NO₂, —N₃, —S(O)R^(d),—S(O)₂R^(d), —S(O)₂OR^(d), —S(O)NR^(c)R^(c); —S(O)₂NR^(c)R^(c),—OS(O)R^(d), —OS(O)₂R^(d), —OS(O)₂OR^(d), —OS(O)NR^(c)R^(c),—OS(O)₂NR^(c)R^(c), —C(O)R^(d), C(O)OR^(d), —C(O)NR^(c)R^(c),—C(NH)NR^(c)R^(c), —OC(O)R^(d), —SC(O)R^(d), —OC(O)OR^(d), —SC(O)OR^(d),—OC(O)NR^(c)R^(c), —SC(O)NR^(c)R^(c), —OC(NH)NR^(c)R^(c),—SC(H)NR^(c)R^(c), —[NHC(O)]_(n)R^(d), —[NHC(O)]_(n)OR^(d),—[NHC(O)]_(n)NR^(c)R^(c) and —[NHC(NH)_(n)]NR^(c)R^(c), (C5-C10) aryloptionally substituted with one or more of the same or different R⁸groups, (C6-C16) arylalkyl optionally substituted with one or more ofthe same or different R⁸ groups, 5-10 membered heteroaryl optionallysubstituted with one or more of the same or different R⁸ groups and 6-16membered heteroarylalkyl optionally substituted with one or more of thesame or different R⁸ groups, (C1-C6) alkyl optionally substituted withone or more of the same or different R⁸ groups, (C1-C4) alkanyloptionally substituted with one or more of the same or different R⁸groups (C2-C4) alkenyl optionally substituted with one or more of thesame or different R⁸ groups and (C2-C4) alkyl optionally substitutedwith one or more of the same or different R⁸ groups; R⁶ independently isselected from the group consisting of hydrogen, —OR^(d), —SR^(d),(C1-C3) haloalkyloxy, (C1-C3) perhaloalkyloxy, —NR^(c)R^(c), halogen,(C1-C3) haloalkyl, (C1-C3) perhaloalkyl, —CN, —NC, —OCN, —SCN, —NO,—NO₂, —N₃—S(O)R^(d), —S(O)₂R^(d), —S(O)₂OR^(d), —S(O)NR^(c)R^(c),—S(O)₂NR^(c)R^(c), —OS(O)R^(d), —OS(O)₂R^(d), —OS(O)₂OR^(d),—OS(O)NR^(c)R^(c), —OS(O)₂NR^(c)R^(c), —C(O)R^(d), —C(O)OR^(d),—C(O)NR^(c)R^(c), —C(NH)NR^(c)R^(c), —OC(O)R^(d), —SC(O)R^(d),OC(O)OR^(d), SC(O)OR^(d), —OC(O)NR^(c)R^(c), —SC(O)NR^(c)R^(c),—OC(NH)NR^(c)R^(c), —SC(NH)NR^(c)R^(c), —[NHC(O)]_(n)R^(d),—[NHC(O)]_(n)OR^(d), —[NHC(O)]_(n)NR^(c)R^(c) and—[NHC(NH)]_(n)NR^(c)R^(c), (C5-C10) aryl optionally substituted with oneor more of the same or different R⁸ groups, (C6-C16) arylalkyloptionally substituted with one or more of the same or different R⁸groups, 5-10 membered heteroaryl optionally substituted with one or moreof the same or different R⁸ groups and 6-16 membered heteroarylalkyloptionally substituted with one or more of the same or different R⁸groups; R⁸ is selected from the group consisting of R^(a), R^(b), R^(a)substituted with one or more of the same or different R^(a) or R^(b),—OR^(a) substituted with one or more of the same or different R^(a) orR^(b), —B(OR^(a))₂, —B(NR^(c)R^(c))₂, —(CH₂)_(m)—R^(b),—(CHR^(a))_(m)—R^(b), —O—(CH₂)_(m)—R^(b), —S—(CH₂)_(m)—R^(b),—O—CHR^(a)R^(b), —O—CR^(a)(R^(b))₂, —O—(CHR^(a))_(m)—R^(b),—O—(CH₂)_(m)—CH[(CH₂)_(m)R^(b)]R^(b), —S—(CHR^(a))_(m)—R^(b),—C(O)NH—(CH₂)_(m)—R^(b), —C(O)NH—(CHR^(a))_(m)—R^(b),—O—(CH₂)_(m)—C(O)NH—(CH₂)_(m)—R^(b),—S—(CH₂)_(m)—C(O)NH—(CH₂)_(m)—R^(b),—O—(CHR^(a))_(m)—C(O)NH—(CHR^(a))_(m)—R^(b),—S—(CHR^(a))_(m)—C(O)NH—(CHR^(a))_(m)—R^(b), —NH—(CH₂)_(m)—R^(b),—NH—(CHR^(a))_(m)—R^(b), —NH[(CH₂)_(m)R^(b)], —N[(CH₂)_(m)R^(b)]₂,—NH—C(O)—NH—(CH₂)_(m)—R^(b), —NH—C(O)—(CH₂)_(m)—CHR^(b)R^(b) and—NH—(CH₂)_(m)—C(O)—NH—(CH₂)_(m)—R^(b); each R³¹ is, independently of theothers, hydrogen or (C1-C6) alkyl optionally substituted with one ormore of the same or different R⁸ groups, each R³⁵ is, independently ofthe other, selected from the group consisting of hydrogen and R⁸, or,alternatively, the two R³⁵ groups are taken together to form an oxo(═O), or ═NR³⁸ group; each R³⁶ is, independently of the others, selectedfrom the group consisting of hydrogen and (C1-C6) alkyl; R³⁸ is selectedfrom the group consisting of hydrogen, (C1-C6) alkyl and (C5-C14) aryl;each R^(a) is, independently of the others, selected from the groupconsisting of hydrogen, (C1-C6) alkyl, (C3-C8) cycloalkyl, (C4-C11)cycloalkylalkyl, (C5-C10) aryl, (C6-C16) arylalkyl, 2-6 memberedheteroalkyl, 3-8 membered cycloheteroalkyl, 11 memberedcycloheteroalkylalkyl, 5-10 membered heteroaryl and 6-16 memberedheteroarylalkyl; each R^(b) is, independently selected from the groupconsisting of ═O, —OR^(d), (C1-C3) haloalkyloxy, ═S, —SR^(d), ═NR^(d),═NOR^(d), —NR^(c)R^(c), halogen, —CF₃, —CN, —NC, —OCN, —SCN, —NO, —NO₂,═N₂, —N₃, —S(O)R^(d), —S(O)₂R^(d), —S(O)₂OR^(d), —S(O)NR^(c)R^(c),—S(O)₂NR^(c)R^(c), —OS(O)R^(d), —OS(O)₂R^(d), —OS(O)₂OR^(d),—OS(O)₂NR^(c)R^(c), —C(O)R^(d), —C(O)OR^(d), —C(O)NR^(c)R^(c),—C(NH)NR^(c)R^(c), —C(NR^(a))NR^(c)R^(c), —C(NOH)R^(a),—C(NOH)NR^(c)R^(c), —OC(O)R^(d), —OC(O)OR^(d), —OC(O)NR^(c)R^(c),—OC(NH)NR^(c)R^(c), —OC(NR^(a))NR^(c)R^(c), —[NHC(O)]_(n)R^(d),—[NR^(a)C(O)]_(n)R^(d), —[NHC(O)]_(n)OR^(d), —[NR^(a)C(O)]_(n)OR^(d),—[NHC(O)]_(n)NR^(c)R^(c), —[NR^(a)C(O)]_(n)R^(c)R^(c),—[NHC(NH)]_(n)NR^(c)R^(c) and —[NR^(a)C(NR^(a))]_(n)NR^(c)R^(c); eachR^(c) is, independently of the others, R^(a), or alternatively, twoR^(c), taken together with the nitrogen atom to which they are bonded,form 5 to 8-membered cycloheteroalkyl or heteroaryl, saidcycloheteroalkyl and heteroaryl each optionally comprising one or moreof the same or different additional heteroatoms and each optionallysubstituted with one or more of the same or different R^(a) or R^(b)groups; each R^(d) is, independently of the others, R^(a); each m is,independently of the others, an integer from 1 to 3; each n is,independently of the others, an integer from 0 to 3; and o is an integerfrom 1 to
 6. 31. The method of claim 30, wherein the autoimmune diseaseis selected from the group consisting of Hashimoto's thyroiditis,autoimmune hemolytic anemia, autoimmune atrophic gastritis of perniciousanemia, autoimmune encephalomyelitis, autoimmune orchitis, Goodpasture'sdisease, autoimmune thrombocytopenia, sympathetic ophthalmia, myastheniagravis, Graves' disease, primary biliary cirrhosis, chronic aggressivehepatitis, ulcerative colitis, membranous glomerulopathy, systemic lupuserythematosis, rheumatoid arthritis, Sjogren's syndrome, Reiter'ssyndrome, polymyositis-dermatomyositis, systemic sclerosis,polyarteritis nodosa, multiple sclerosis and bullous pemphigoid. Para 29on pages 13-14, para 154 on pages 58-59
 32. The method of claim 31,wherein the autoimmune disease is selected from the group consisting ofHashimoto's thyroiditis, autoimmune hemolytic anemia, autoimmuneatrophic gastritis of pernicious anemia, autoimmune encephalomyelitis,autoimmune orchitis, Goodpasture's disease, autoimmune thrombocytopenia,sympathetic ophthalmia, myasthenia gravis, Graves' disease, primarybiliary cirrhosis, chronic aggressive hepatitis, ulcerative colitis, andmembranous glomerulopathy.
 33. The method of claim 31, wherein theautoimmune disease is selected from the group consisting of systemiclupus erythematosis, rheumatoid arthritis, Sjogren's syndrome, Reiter'ssyndrome, polymyositis-dermatomyositis, systemic sclerosis,polyarteritis nodosa, multiple sclerosis and bullous pemphigoid.
 34. Themethod of claim 33, wherein the autoimmune disease is systemic lupuserythematosis.
 35. The method of claim 33, wherein the autoimmunedisease is rheumatoid arthritis.
 36. The method of claim 33, wherein theautoimmune disease is multiple sclerosis.